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The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported., Competing Interests: Competing interest statement: J.W.P., A.R.H., and H.S. have a financial interest in Fluxus Inc., which is developing optofluidic devices., (Copyright © 2021 the Author(s). Published by PNAS.)

A microfluidic sample preparation multiplexer (SPM) and assay procedure is developed to improve amplification-free detection of Ebola virus RNA from blood. While a previous prototype successfully detected viral RNA following off-chip RNA extraction from infected cells, the new device and protocol can detect Ebola virus in raw blood with clinically relevant sensitivity. The Ebola RNA is hybridized with sequence specific capture and labeling DNA probes in solution and then the complex is pulled down onto capture beads for purification and concentration. After washing, the captured RNA target is released by irradiating the photocleavable DNA capture probe with ultraviolet (UV) light. The released, labeled, and purified RNA is detected by a sensitive and compact fluorometer. Exploiting these capabilities, a detection limit of 800 attomolar (aM) is achieved without target amplification. The new SPM can run up to 80 assays in parallel using a pneumatic multiplexing architecture. Importantly, our new protocol does not require time-consuming and problematic off-chip probe conjugation and washing. This improved SPM and labeling protocol is an important step toward a useful POC device and assay.

Simultaneous detection of multiple pathogens and samples (multiplexing) is one of the key requirements for diagnostic tests in order to enable fast, accurate and differentiated diagnoses. Here, we introduce a novel, highly scalable, photonic approach to multiplex analysis with single virus sensitivity. A solid-core multimode interference (MMI) waveguide crosses multiple fluidic waveguide channels on an optofluidic chip to create multi-spot excitation patterns that depend on both the wavelength and location of the channel along the length of the MMI waveguide. In this way, joint spectral and spatial multiplexing is implemented that encodes both spatial and spectral information in the time dependent fluorescence signal. We demonstrate this principle by using two excitation wavelengths and three fluidic channels to implement a 6x multiplex assay with single virus sensitivity. High fidelity detection and identification of six different viruses from a standard influenza panel is reported. This multimodal multiplexing strategy scales favorably to large numbers of targets or large numbers of clinical samples. Further, since single particles are detected unbound in flow, the technique can be broadly applied to direct detection of any fluorescent target, including nucleic acids and proteins.

Dynamic reconfiguration of photonic function is one of the hallmarks of optofluidics. A number of approaches have been taken to implement optical tunability in microfluidic devices. However, a device architecture that allows for simultaneous high-performance microfluidic fluid handling as well as dynamic optical tuning has not been demonstrated. Here, we introduce such a platform based on a combination of solid- and liquid-core polydimethylsiloxane (PDMS) waveguides that also provides fully functioning microvalve-based sample handling. A combination of these waveguides forms a liquid-core multimode interference waveguide that allows for multi-modal tuning of waveguide properties through core liquids and pressure/deformation. We also introduce a novel lifting-gate lightvalve that simultaneously acts as a fluidic microvalve and optical waveguide, enabling mechanically reconfigurable light and fluid paths and seamless incorporation of controlled particle analysis. These new functionalities are demonstrated by an optical switch with >45 dB extinction ratio and an actuatable particle trap for analysis of biological micro- and nanoparticles.

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context--the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine.

Single λ-DNA molecules are detected on a nanopore-gated optofluidic chip electrically and optically. Statistical variations in the single particle trajectories are used to predict the intensity distribution of the fluorescence signals.

The analysis of individual biological nanoparticles has significantly advanced our understanding of fundamental biological processes but is also rapidly becoming relevant for molecular diagnostic applications in the emerging field of personalized medicine. Both optical and electrical methods for the detection and analysis of single biomolecules have been developed, but they are generally not used in concert and in suitably integrated form to allow for multimodal analysis with high throughput. Here we report on a dual-mode electrical and optical single-nanoparticle sensing device with capabilities that would not be available with each technique individually. The new method is based on an optofluidic chip with an integrated nanopore that serves as a smart gate to control the delivery of individual nanoparticles to an optical excitation region for ensemble-free optical analysis in rapid succession. We demonstrate electro-optofluidic size discrimination of fluorescent nanobeads, electro-optical detection of single fluorescently labeled influenza viruses, and the identification of single viruses within a mixture of equally sized fluorescent nanoparticles with up to 100% fidelity.

We report size-based sorting of micro- and sub-micron particles using optical forces on a planar optofluidic chip. Two different combinations of fluid flow and optical beam directions in liquid-core waveguides are demonstrated. These methods allow for tunability of size selection and sorting with efficiencies as high as 100%. Very good agreement between experimental results and calculated particle trajectories in the presence of flow and optical forces is found.

Complete integration of microfluidic and optical functions in a single lab-on-chip device is one goal of optofluidics. Here, we demonstrate the hybrid integration of a PDMS-based fluid handling layer with a silicon-based optical detection layer in a single optofluidic system. The optical layer consists of a liquid-core antiresonant reflecting optical waveguide (ARROW) chip that is capable of single particle detection and interfacing with optical fiber. Integrated devices are reconfigurable and able to sustain high pressures despite the small dimensions of the liquid-core waveguide channels. We show the combination of salient sample preparation capabilities-particle mixing, distribution, and filtering-with single particle fluorescence detection. Specifically, we demonstrate fluorescent labelling of λ-DNA, followed by flow-based single-molecule detection on a single device. This points the way towards amplification-free detection of nucleic acids with low-complexity biological sample preparation on a chip.

Liquid filled waveguides that form the basis for on-chip biophotonics diagnostic platforms have primarily found application in fluorescence and Raman spectroscopy experiments that require sensitive discrimination between weak analyte signals and a variety of background signals. Primary sources of background signal can include light from excitation sources (strong, narrow frequency band) and photoluminescence generated in waveguide cladding layers (weak, wide frequency band). Here we review both solid and liquid core filtering structures which are based on anti-resonant reflection that can be integrated with waveguides for attenuating undesirable optical bands. Important criteria to consider for an optimized biosensor include cladding layer materials that minimize broad-spectrum photoluminescence and optimize layer thicknesses for creating a desired spectral response in both solid and liquid guiding layers, and a microfabrication process capable of producing regions with variable spectral response. New results describing how spurious fluorescence can be minimized by optimized thermal growth conditions and how liquid-core filter discrimination can be tuned with liquid core waveguide length are presented., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe analysis and control of 50S ribosomal subunits by a solid-state 45nm diameter nanopore incorporated in a microfluidic chip. When used as a resistive pulse sensor, translocation of single 50S subunits through the nanopore produces current blockades that have a linear dependence on applied voltage. Introduction of individual subunits into the fluidic channel shows a threshold behavior that allows controlled entry of individual 50S ribosomal subunits. The incorporation of nanopores into a larger optofluidic chip system opens possibilities for electrical and optical studies of single ribosomes in well-defined and rapidly variable chemical environments., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Spectral filtering is an essential component of biophotonic methods such as fluorescence and Raman spectroscopy. Predominantly utilized in bulk microscopy, filters require efficient and selective transmission or removal of signals at one or more wavelength bands. However, towards highly sensitive and fully self-contained lab-on-chip systems, the integration of spectral filters is an essential step. In this work, a novel optofluidic solution is presented in which a liquid-core optical waveguide both transports sample analytes and acts as an efficient filter for advanced spectroscopy. To this end, the wavelength dependent nature of interference-based antiresonant reflecting optical waveguide technology is exploited. An extinction of 37 dB, a narrow rejection band of only 2.5 nm and a free spectral range of 76 nm using three specifically designed dielectric layers are demonstrated. These parameters result in an 18.4-fold increase in the signal-to-noise ratio for on-chip fluorescence detection. In addition, liquid-core waveguide filters with three operating wavelengths were designed for Förster resonance energy transfer detection and demonstrated using doubly labeled oligonucleotides. Incorporation of high-performance spectral processing illustrates the power of the optofluidic concept where fluidic channels also perform optical functions to create innovative and highly integrated lab-on-chip devices.

A new waveguide design for an optofluidic chip is presented. It mitigates multi-mode behavior in solid and liquid-core waveguides by increasing fundamental mode coupling to 82% and 95%, respectively. Additionally, we demonstrate a six-fold improvement in lateral confinement of optically guided dielectric microparticles and double the detection efficiency of fluorescent particles.

We present a fully planar integrated optofluidic platform that permits single particle detection, manipulation and analysis on a chip. Liquid-core optical waveguides guide both light and fluids in the same volume. They are integrated with fluidic reservoirs and solid-core optical waveguides to define sub-picoliter excitation volumes and collect the optical signal, resulting in fully planar beam geometries. Single fluorescently labeled liposomes are used to demonstrate the capabilities of the optofluidic chip. Liposome motion is controlled electrically, and fluorescence correlation spectroscopy (FCS) is used to determine concentration and dynamic properties such as diffusion coefficient and velocity. This demonstration of fully planar particle analysis on a semiconductor chip may lead to a new class of planar optofluidics-based instruments.

A microfluidic sample preparation multiplexer (SPM) and assay procedure is developed to improve amplification-free detection of Ebola virus RNA from blood. While a previous prototype successfully detected viral RNA following off-chip RNA extraction from infected cells, the new device and protocol can detect Ebola virus in raw blood with clinically relevant sensitivity. The Ebola RNA is hybridized with sequence specific capture and labeling DNA probes in solution and then the complex is pulled down onto capture beads for purification and concentration. After washing, the captured RNA target is released by irradiating the photocleavable DNA capture probe with ultraviolet (UV) light- The released, labeled, and purified RNA is detected by a sensitive and compact fluorometer. Exploiting these capabilities, a detection limit of 800 attomolar (aM) is achieved without target amplification. The new SPM can run up to 80 assays in parallel using a pneumatic multiplexing architecture. Importantly, our new protocol does not require time-consuming and problematic off-chip probe conjugation and washing. This improved SPM and labeling protocol is an important step toward a useful POC device and assay. [ABSTRACT FROM AUTHOR]