Design Principles of DNA-Barcodes for Nanopore-FET Readout, Based on Molecular Dynamics and TCAD Simulations.

Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s-1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s-1, with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.

On-chip flow cytometer using integrated photonics for the detection of human leukocytes.

Differentiation between leukocyte subtypes like monocytes and lymphocytes is essential for cell therapy and research applications. To guarantee the cost-effective delivery of functional cells in cell therapies, billions of cells must be processed in a limited time. Yet, the sorting rates of commercial cell sorters are not high enough to reach the required yield. Process parallelization by using multiple instruments increases variability and production cost. A compact solution with higher throughput can be provided by multichannel flow cytometers combining fluidics and optics on-chip. In this work, we present a micro-flow cytometer with monolithically integrated photonics and fluidics and demonstrate that both the illumination of cells, as well as the collection of scattered light, can be realized using photonic integrated circuits. Our device is the first with sufficient resolution for the discrimination of lymphocytes and monocytes. Innovations in microfabrication have enabled complete integration of miniaturized photonic components and fluidics in a CMOS-compatible wafer stack. In combination with external optics, the device is ready for the collection of fluorescence using the on-chip excitation.

Inverse design assisted coherent optical lattices.

We explore the use of inverse design methods for the generation of periodic optical patterns in photonic integrated circuits. A carefully selected objective function based on the integer lattice method, which is an algebraic technique for optical lattice generation, is shown to be key for successful device design. Furthermore, we present a polychromatic pattern generating device that switches between optical lattices with different symmetry and periodicity depending on the operating wavelength. Important links are drawn between optical coherent lattices and optical potentials, pointing towards practical applications in the fields of quantum simulations and computing, optical trapping, and bio-sensing.

Analysis tools for single-monomer measurements of self-assembly processes.

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin.

Performance and limitation estimation of a three-tap gated imaging sensor in wide field time-gated fluorescence lifetime imaging systems.

In this paper, a computational performance analysis is presented of a wide-field time-gated fluorescence lifetime imaging microscope (FLIM) using practically realizable properties of the laser, sample, and a three-tap time-gated CMOS image sensor. The impact of these component-level properties on the accuracy and the precision of the measurement results are estimated and discussed based on Monte Carlo simulations. The correlation between the detector speed and the accuracy of the extracted fluorescence lifetime is studied, and the minimum required incident photoelectron number of each pixel is estimated for different detector speeds and different fluorescence lifetime measurements. In addition, the detection limits due to the dark current and the parasitic light sensitivity of the detector are also investigated. This work gives an overview of the required fluorescence emission condition as well as the required detector properties for a three-tap time-gated image sensor to achieve good FLIM data in biological applications.

Revival and Expansion of the Theory of Coherent Lattices.

An effective way to design structured coherent wave interference patterns that builds on the theory of coherent lattices, is presented. The technique combines prime number factorization in the complex plane with moiré theory to provide a robust way to design structured patterns with variable spacing of intensity maxima. In addition, the proposed theoretical framework facilitates an elegant computation of previously unexplored high-order superlattices both for the periodic and quasiperiodic case. A number of beam configurations highlighting prime examples of patterns for lattices with three-, four-, and fivefold symmetry are verified in a multibeam interference experiment.

Accurate modeling of a biological nanopore with an extended continuum framework.

Despite the broad success of biological nanopores as powerful instruments for the analysis of proteins and nucleic acids at the single-molecule level, a fast simulation methodology to accurately model their nanofluidic properties is currently unavailable. This limits the rational engineering of nanopore traits and makes the unambiguous interpretation of experimental results challenging. Here, we present a continuum approach that can faithfully reproduce the experimentally measured ionic conductance of the biological nanopore Cytolysin A (ClyA) over a wide range of ionic strengths and bias potentials. Our model consists of the extended Poisson-Nernst-Planck and Navier-Stokes (ePNP-NS) equations and a computationally efficient 2D-axisymmetric representation for the geometry and charge distribution of the nanopore. Importantly, the ePNP-NS equations achieve this accuracy by self-consistently considering the finite size of the ions and the influence of both the ionic strength and the nanoscopic scale of the pore on the local properties of the electrolyte. These comprise the mobility and diffusivity of the ions, and the density, viscosity and relative permittivity of the solvent. Crucially, by applying our methodology to ClyA, a biological nanopore used for single-molecule enzymology studies, we could directly quantify several nanofluidic characteristics difficult to determine experimentally. These include the ion selectivity, the ion concentration distributions, the electrostatic potential landscape, the magnitude of the electro-osmotic flow field, and the internal pressure distribution. Hence, this work provides a means to obtain fundamental new insights into the nanofluidic properties of biological nanopores and paves the way towards their rational engineering.

Electro-Osmotic Vortices Promote the Capture of Folded Proteins by PlyAB Nanopores.

Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76-81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores.

Engineering and Modeling the Electrophoretic Trapping of a Single Protein Inside a Nanopore.

The ability to confine and to study single molecules has enabled important advances in natural and applied sciences. Recently, we have shown that unlabeled proteins can be confined inside the biological nanopore Cytolysin A (ClyA) and conformational changes monitored by ionic current recordings. However, trapping small proteins remains a challenge. Here, we describe a system where steric, electrostatic, electrophoretic, and electro-osmotic forces are exploited to immobilize a small protein, dihydrofolate reductase (DHFR), inside ClyA. Assisted by electrostatic simulations, we show that the dwell time of DHFR inside ClyA can be increased by orders of magnitude (from milliseconds to seconds) by manipulation of the DHFR charge distribution. Further, we describe a physical model that includes a double energy barrier and the main electrophoretic components for trapping DHFR inside the nanopore. Simultaneous fits to the voltage dependence of the dwell times allowed direct estimates of the cis and trans translocation probabilities, the mean dwell time, and the force exerted by the electro-osmotic flow on the protein (≅9 pN at -50 mV) to be retrieved. The observed binding of NADPH to the trapped DHFR molecules suggested that the engineered proteins remained folded and functional inside ClyA. Contact-free confinement of single proteins inside nanopores can be employed for the manipulation and localized delivery of individual proteins and will have further applications in single-molecule analyte sensing and enzymology studies.

Two-Photon Microscopy with a Double-Wavelength Metasurface Objective Lens.

Two-photon microscopy is a key imaging technique in life sciences due to its superior deep-tissue imaging capabilities. Light-weight and compact two-photon microscopes are of great interest because of their applications for in vivo deep brain imaging. Recently, dielectric metasurfaces have enabled a new category of small and lightweight optical elements, including objective lenses. Here we experimentally demonstrate two-photon microscopy using a double-wavelength metasurface lens. It is specifically designed to focus 820 and 605 nm light, corresponding to the excitation and emission wavelengths of the measured fluorophors, to the same focal distance. The captured two-photon images are qualitatively comparable to the ones taken by a conventional objective lens. Our metasurface lens can enable ultracompact two-photon microscopes with similar performance compared to current systems that are usually based on graded-index-lenses. In addition, further development of tunable metasurface lenses will enable fast axial scanning for volumetric imaging.

High spatial resolution nanoslit SERS for single-molecule nucleobase sensing.

Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS.

Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy.

Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i.e., signal to background ratio (SBR), is still lacking. In this Letter, we present a novel technique that allows to discriminate the emission patterns of free and bound fluorophores simultaneously by collecting both angular and spectral information. The spectrum was probed at multiple positions in the back focal plane using a multimode fiber connected to a spectrometer and the difference in intensity between two fluorophores was used to calculate the SBR. Our study clearly reveals that increasing the angle of SAF collection enhances the surface sensitivity, albeit at the cost of decreased signal intensity. Furthermore, our findings are fully supported by full-field 3D simulations.

Near-Field Mapping of Optical Fabry-Perot Modes in All-Dielectric Nanoantennas.

Subwavelength optical resonators and scatterers are dramatically expanding the toolset of the optical sciences and photonics engineering. By offering the opportunity to control and shape light waves in nanoscale volumes, recent developments using high-refractive-index dielectric scatterers gave rise to efficient flat-optical components such as lenses, polarizers, phase plates, color routers, and nonlinear elements with a subwavelength thickness. In this work, we take a deeper look into the unique interaction of light with rod-shaped amorphous silicon scatterers by tapping into their resonant modes with a localized subwavelength light source-an aperture scanning near-field probe. Our experimental configuration essentially constitutes a dielectric antenna that is locally driven by the aperture probe. We show how leaky transverse electric and magnetic modes can selectively be excited and form specific near-field distribution depending on wavelength and antenna dimensions. The probe's transmittance is furthermore enhanced upon coupling to the Fabry-Perot cavity modes, revealing all-dielectric nanorods as efficient transmitter antennas for the radiation of subwavelength emitters, in addition to constituting an elementary building block for all-dielectric metasurfaces and flat optics.

Electrically Driven Unidirectional Optical Nanoantennas.

Directional antennas revolutionized modern day telecommunication by enabling precise beaming of radio and microwave signals with minimal loss of energy. Similarly, directional optical nanoantennas are expected to pave the way toward on-chip wireless communication and information processing. Currently, on-chip integration of such antennas is hampered by their multielement design or the requirement of complicated excitation schemes. Here, we experimentally demonstrate electrical driving of in-plane tunneling nanoantennas to achieve broadband unidirectional emission of light. Far-field interference, as a result of the spectral overlap between the dipolar emission of the tunnel junction and the fundamental quadrupole-like resonance of the nanoantenna, gives rise to a directional radiation pattern. By tuning this overlap using the applied voltage, we record directivities as high as 5 dB. In addition to electrical tunability, we also demonstrate passive tunability of the directivity using the antenna geometry. These fully configurable electrically driven nanoantennas provide a simple way to direct optical energy on-chip using an extremely small device footprint.

Development of nanostars as a biocompatible tumor contrast agent: toward in vivo SERS imaging.

The need for sensitive imaging techniques to detect tumor cells is an important issue in cancer diagnosis and therapy. Surface-enhanced Raman scattering (SERS), realized by chemisorption of compounds suitable for Raman spectroscopy onto gold nanoparticles, is a new method for detecting a tumor. As a proof of concept, we studied the use of biocompatible gold nanostars as sensitive SERS contrast agents targeting an ovarian cancer cell line (SKOV3). Due to a high intracellular uptake of gold nanostars after 6 hours of exposure, they could be detected and located with SERS. Using these nanostars for passive targeting after systemic injection in a xenograft mouse model, a detectable signal was measured in the tumor and liver in vivo. These signals were confirmed by ex vivo SERS measurements and darkfield microscopy. In this study, we established SERS nanostars as a highly sensitive contrast agent for tumor detection, which opens the potential for their use as a theranostic agent against cancer.

Asymmetric plasmonic induced ionic noise in metallic nanopores.

We present distinct asymmetric plasmon-induced noise properties of ionic transport observed through gold coated nanopores. We thoroughly investigated the effects of bias voltage and laser illumination. We show that the potential drop across top-coated silicon nanocavity pores can give rise to a large noise asymmetry (∼2-3 orders of magnitude). Varying the bias voltage has an appreciable effect on the noise density spectra, typically in the Lorentzian components. The laser power is found to strongly affect the ionic noise level as well as the voltage threshold for light-induced noise generation. The asymmetric noise phenomenon is attributed to plasmon-induced interfacial reactions which promote light-induced charge fluctuation in the ion flow and allow voltage modulation of photo-induced carriers surmounting over such Schottky junctions. We further compare the ionic noise performances of gold nanocavities containing different material stacks, among which thermal oxide passivation of the silicon successfully mitigates the light-induced noise and is also fully CMOS-compatible. The understanding of the described noise characteristics will help to foster multiple applications using related structures including plasmonic-based sensing or plasmon-induced catalysis such as water splitting or solar energy conversion devices.

All-Dielectric Antenna Wavelength Router with Bidirectional Scattering of Visible Light.

An optical antenna forms the subwavelength bridge between free space optical radiation and localized electromagnetic energy. Its localized electromagnetic modes strongly depend on its geometry and material composition. Here, we present the design and experimental realization of a novel V-shaped all-dielectric antenna based on high-index amorphous silicon with a strong magnetic dipole resonance in the visible range. As a result, it exhibits extraordinary bidirectional scattering into diametrically opposite directions. The scattering direction is effectively controlled by the incident wavelength, rendering the antenna a passive bidirectional wavelength router. A detailed multipole decomposition analysis reveals that the excitation and abrupt phase change of an out-of-plane polarized magnetic dipole and an in-plane electric quadrupole are essential for the directivity switching. Previously, noble metals have been extensively exploited for plasmonic directional nanoantenna design. However, these inevitably suffer from high intrinsic ohmic losses and a relatively weak magnetic response to the incident light. Compared to a similar gold plasmonic nanoantenna design, we show that the silicon-based antennas demonstrate stronger magnetic scattering with minimal absorption losses. Our results indicate that all-dielectric antennas will open exciting possibilities for efficient manipulation of light-matter interactions.

Direct Fabrication of Monodisperse Silica Nanorings from Hollow Spheres - A Template for Core-Shell Nanorings.

We report a new type of nanosphere colloidal lithography to directly fabricate monodisperse silica (SiO2) nanorings by means of reactive ion etching of hollow SiO2 spheres. Detailed TEM, SEM, and AFM structural analysis is complemented by a model describing the geometrical transition from hollow sphere to ring during the etching process. The resulting silica nanorings can be readily redispersed in solution and subsequently serve as universal templates for the synthesis of ring-shaped core-shell nanostructures. As an example we used silica nanorings (with diameter of ∼200 nm) to create a novel plasmonic nanoparticle topology, a silica-Au core-shell nanoring, by self-assembly of Au nanoparticles (<20 nm) on the ring's surface. Spectroscopic measurements and finite difference time domain simulations reveal high quality factor multipolar and antibonding surface plasmon resonances in the near-infrared. By loading different types of nanoparticles on the silica core, hybrid and multifunctional composite nanoring structures could be realized for applications such as MRI contrast enhancement, catalysis, drug delivery, plasmonic and magnetic hyperthermia, photoacoustic imaging, and biochemical sensing.

Raman fingerprinting of single dielectric nanoparticles in plasmonic nanopores.

Plasmonic nano-apertures are commonly used for the detection of small particles such as nanoparticles and proteins by exploiting electrical and optical techniques. Plasmonic nanopores are metallic nano-apertures sitting on a thin membrane with a tiny hole. It has been shown that plasmonic nanopores with a given geometry identify internal molecules using Surface Enhanced Raman Spectroscopy (SERS). However, label-free identification of a single dielectric nanoparticle requires a highly localized field comparable to the size of the particle. Additionally, the particle's Brownian motion can jeopardize the amount of photons collected from a single particle. Here, we demonstrate that the combination of optical trapping and SERS can be used for the detection and identification of 20 nm polystyrene nanoparticles in plasmonic nanopores. This work is anticipated to contribute to the detection of small bioparticles, optical trapping and nanotribology studies.

Bright and dark plasmon resonances of nanoplasmonic antennas evanescently coupled with a silicon nitride waveguide.

In this work we investigate numerically and experimentally the resonance wavelength tuning of different nanoplasmonic antennas excited through the evanescent field of a single mode silicon nitride waveguide and study their interaction with this excitation field. Experimental interaction efficiencies up to 19% are reported and it is shown that the waveguide geometry can be tuned in order to optimize this interaction. Apart from the excitation of bright plasmon modes, an efficient coupling between the evanescent field and a dark plasmonic resonance is experimentally demonstrated and theoretically explained as a result of the propagation induced phase delay.