Digital microarrays: single-molecule readout with interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology’s Achilles’ heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule (“digital”) regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform’s primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique’s simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.The authors wish to thank Oguzhan Avci and Jacob Trueb for thoughtful comments and suggestions regarding numerical optimization of the optical system. This work was funded in part by a research contract with ASELSAN, Inc. and the Wallace H. Coulter Foundation 2010 Coulter Translational Award. (ASELSAN, Inc.; Wallace H. Coulter Foundation Coulter Translational Award)Accepted manuscrip

Nanoparticle Classification in Wide-field Interferometric Microscopy by Supervised Learning from Model

Interference enhanced wide-field nanoparticle imaging is a highly sensitive technique that has found numerous applications in labeled and label-free sub-diffraction-limited pathogen detection. It also provides unique opportunities for nanoparticle classification upon detection. More specif- ically, the nanoparticle defocus images result in a particle-specific response that can be of great utility for nanoparticle classification, particularly based on type and size. In this work, we com- bine a model based supervised learning algorithm with a wide-field common-path interferometric microscopy method to achieve accurate nanoparticle classification. We verify our classification schemes experimentally by using gold and polystyrene nanospheres.Comment: 5 pages, 2 figure

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.First author draf

A digital microarray using interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and dynamic range of traditional fluorescence microarrays compared to other techniques have been the technology's Achilles' Heel, and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ('digital') regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about three orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10x objective lens. This approach does not require any chemical enhancement such as silver deposition, and scans arrays with a throughput similar to commercial fluorescence devices. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about one million directly from a single scan

A digital microarray using interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and dynamic range of traditional fluorescence microarrays compared to other techniques have been the technology's Achilles' Heel, and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ('digital') regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about three orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10x objective lens. This approach does not require any chemical enhancement such as silver deposition, and scans arrays with a throughput similar to commercial fluorescence devices. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about one million directly from a single scan.First author draf

Interferometric detection and enumeration of viral particles using Si-based microfluidics

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.This work was supported in part by a research contract with the ASELSAN Research Center, Ankara, Turkey, and in part by the European Union's Horizon 2020 FET Open program under Grant 766466-INDEX. (ASELSAN Research Center, Ankara, Turkey; 766466-INDEX - European Union's Horizon 2020 FET Open program)First author draf

Rapid mapping of digital integrated circuit logic gates via multi-spectral backside imaging

Modern semiconductor integrated circuits are increasingly fabricated at untrusted third party foundries. There now exist myriad security threats of malicious tampering at the hardware level and hence a clear and pressing need for new tools that enable rapid, robust and low-cost validation of circuit layouts. Optical backside imaging offers an attractive platform, but its limited resolution and throughput cannot cope with the nanoscale sizes of modern circuitry and the need to image over a large area. We propose and demonstrate a multi-spectral imaging approach to overcome these obstacles by identifying key circuit elements on the basis of their spectral response. This obviates the need to directly image the nanoscale components that define them, thereby relaxing resolution and spatial sampling requirements by 1 and 2 - 4 orders of magnitude respectively. Our results directly address critical security needs in the integrated circuit supply chain and highlight the potential of spectroscopic techniques to address fundamental resolution obstacles caused by the need to image ever shrinking feature sizes in semiconductor integrated circuits

Exciton mediated one phonon resonant Raman scattering from one-dimensional systems

We use the Kramers-Heisenberg approach to derive a general expression for the resonant Raman scattering cross section from a one-dimensional (1D) system explicitly accounting for excitonic effects. The result should prove useful for analyzing the Raman resonance excitation profile lineshapes for a variety of 1D systems including carbon nanotubes and semiconductor quantum wires. We apply this formalism to a simple 1D model system to illustrate the similarities and differences between the free electron and correlated electron-hole theories.Comment: 10 pages, 6 figure

Interactive Sketching of Mannequin Poses

It can be easy and even fun to sketch humans in different poses. In contrast, creating those same poses on a 3D graphics 'mannequin' is comparatively tedious. Yet 3D body poses are necessary for various downstream applications. We seek to preserve the convenience of 2D sketching while giving users of different skill levels the flexibility to accurately and more quickly pose/refine a 3D mannequin. At the core of the interactive system, we propose a machine-learning model for inferring the 3D pose of a CG mannequin from sketches of humans drawn in a cylinder-person style. Training such a model is challenging because of artist variability, a lack of sketch training data with corresponding ground truth 3D poses, and the high dimensionality of human pose-space. Our unique approach to synthesizing vector graphics training data underpins our integrated ML-and-kinematics system. We validate the system by tightly coupling it with a user interface, and by performing a user study, in addition to quantitative comparisons

Controlling the charge transfer flow at the graphene/pyrene-nitrilotriacetic acid interface

The fabrication of highly efficient bio-organic nanoelectronic devices is still a challenge due to the difficulty in interfacing the biomolecular component to the organic counterparts. One of the ways to overcome this bottleneck is to add a self-assembled monolayer (SAM) in between the electrode and the biological material. The addition of a pyrene-nitrilotriacetic acid layer to a graphene metal electrode enhances the charge transfer within the device. Our theoretical calculations and electrochemical results show that the formation of a pyrene-nitrilotriacetic acid SAM enforces a direct electron transfer from graphene to the SAM, while the addition of the Ni2+ cation and imidazole reverses the charge transfer direction, allowing an atomic control of the electron flow, which is essential for a true working device. © 2018 The Royal Society of Chemistry