Machine learning at the edge for AI-enabled multiplexed pathogen detection.

Multiplexed detection of biomarkers in real-time is crucial for sensitive and accurate diagnosis at the point of use. This scenario poses tremendous challenges for detection and identification of signals of varying shape and quality at the edge of the signal-to-noise limit. Here, we demonstrate a robust target identification scheme that utilizes a Deep Neural Network (DNN) for multiplex detection of single particles and molecular biomarkers. The model combines fast wavelet particle detection with Short-Time Fourier Transform analysis, followed by DNN identification on an AI-specific edge device (Google Coral Dev board). The approach is validated using multi-spot optical excitation of Klebsiella Pneumoniae bacterial nucleic acids flowing through an optofluidic waveguide chip that produces fluorescence signals of varying amplitude, duration, and quality. Amplification-free 3× multiplexing in real-time is demonstrated with excellent specificity, sensitivity, and a classification accuracy of 99.8%. These results show that a minimalistic DNN design optimized for mobile devices provides a robust framework for accurate pathogen detection using compact, low-cost diagnostic devices.

Fast custom wavelet analysis technique for single molecule detection and identification.

Many sensors operate by detecting and identifying individual events in a time-dependent signal which is challenging if signals are weak and background noise is present. We introduce a powerful, fast, and robust signal analysis technique based on a massively parallel continuous wavelet transform (CWT) algorithm. The superiority of this approach is demonstrated with fluorescence signals from a chip-based, optofluidic single particle sensor. The technique is more accurate than simple peak-finding algorithms and several orders of magnitude faster than existing CWT methods, allowing for real-time data analysis during sensing for the first time. Performance is further increased by applying a custom wavelet to multi-peak signals as demonstrated using amplification-free detection of single bacterial DNAs. A 4x increase in detection rate, a 6x improved error rate, and the ability for extraction of experimental parameters are demonstrated. This cluster-based CWT analysis will enable high-performance, real-time sensing when signal-to-noise is hardware limited, for instance with low-cost sensors in point of care environments.

Optical trapping assisted label-free and amplification-free detection of SARS-CoV-2 RNAs with an optofluidic nanopore sensor.

Ultrasensitive, versatile sensors for molecular biomarkers are a critical component of disease diagnostics and personalized medicine as the COVID-19 pandemic has revealed in dramatic fashion. Integrated electrical nanopore sensors can fill this need via label-free, direct detection of individual biomolecules, but a fully functional device for clinical sample analysis has yet to be developed. Here, we report amplification-free detection of SARS-CoV-2 RNAs with single molecule sensitivity from clinical nasopharyngeal swab samples on an electro-optofluidic chip. The device relies on optically assisted delivery of target carrying microbeads to the nanopore for single RNA detection after release. A sensing rate enhancement of over 2,000x with favorable scaling towards lower concentrations is demonstrated. The combination of target specificity, chip-scale integration and rapid detection ensures the practicality of this approach for COVID-19 diagnosis over the entire clinically relevant concentration range from 104-109 copies/mL.

Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.

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.

Optofluidic Amplification-free Multiplex Detection of Viral Hemorrhagic Fevers.

Infectious disease outbreaks such as Ebola and other Viral Hemorrhagic Fevers (VHF) require low-complexity, specific, and differentiated diagnostics as illustrated by the recent outbreak in the Democratic Republic of Congo. Here, we describe amplification-free spectrally multiplex detection of four different VHF total RNA samples using multi-spot excitation on a multimode interference waveguide platform along with combinatorial fluorescence labeling of target nucleic acids. In these experiments, we observed an average of 8-fold greater fluorescence signal amplitudes for the Ebola total RNA sample compared to three other total RNA samples: Lake Victoria Marburg Virus, Ravn Marburg Virus, and Crimean-Congo Hemorrhagic Fever. We have attributed this amplitude amplification to an increased amount of RNA during synthesis of soluble glycoprotein in infection. This hypothesis is confirmed by single molecule detection of the total RNA sample after heat-activated release from the carrier microbeads. From these experiments, we observed at least a 5.3x higher RNA mass loading on the Ebola carrier microbeads compared to the Lake Victoria Marburg carrier microbeads, which is consistent with the known production of soluble glycoprotein during infection.

Optofluidic detection of Zika nucleic acid and protein biomarkers using multimode interference multiplexing.

The recent massive Zika virus (ZIKV) outbreak illustrates the need for rapid and specific diagnostic techniques. Detecting ZIKV in biological samples poses unique problems: antibody detection of ZIKV is insufficient due to cross-reactivity of Zika antibodies with other flaviviruses, and nucleic acid and protein biomarkers for ZIKV are detectable at different stages of infection. Here, we describe a new optofluidic approach for the parallel detection of different molecular biomarkers using multimode interference (MMI) waveguides. We report differentiated, multiplex detection of both ZIKV biomarker types using multi-spot excitation at two visible wavelengths with over 98% fidelity by combining several analysis techniques.

Optimized ARROW-Based MMI Waveguides for High Fidelity Excitation Patterns for Optofluidic Multiplexing.

Multimode interference (MMI) waveguides can be used for multiplexing and de-multiplexing optical signals. High fidelity, wavelength dependent multi-spot patterns from MMI waveguides are useful for sensitive and simultaneous identification of multiple targets in multiplexed fluorescence optofluidic biosensors. Through experiments and simulation, this paper explores design parameters for an MMI rib anti-resonant reflecting optical waveguide (ARROW) in order to produce high fidelity spot patterns at the liquid core biomarker excitation region. Width and etch depth of the single excitation rib waveguide used to excite the MMI waveguide are especially critical because they determine the size of the input optical mode which is imaged at the MMI waveguide's output. To increase optical throughput into the MMI waveguide when light is coupled in from an optical fiber, tapers in the waveguide width can be used for better mode matching.

Buried Rib SiO2 Multimode Interference Waveguides for Optofluidic Multiplexing.

Multimode interference (MMI) waveguides can be used to create wavelength-dependent spot patterns which enables simultaneous analyte detection on a single optofluidic chip, useful for disease diagnostics. The fidelity of such multi-spot patterns is important for high sensitivity and accurate target identification. Buried rib structures have been incorporated into these SiO2-based waveguides to improve environmental stability. Through experiments and simulation, this letter explores design parameters for a buried MMI rib waveguide based on anti-resonant reflecting optical waveguides in order to produce high-fidelity spot patterns. Optimal rib heights and widths are reported in the context of available microfabrication etch technology and performance for an optimized biosensor is shown.

Scalable Spatial-Spectral Multiplexing of Single-Virus Detection Using Multimode Interference Waveguides.

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.

Signal-to-noise Enhancement in Optical Detection of Single Viruses with Multi-spot Excitation.

We present fluorescence detection of single H1N1 viruses with enhanced signal to noise ratio (SNR) achieved by multi-spot excitation in liquid-core anti-resonant reflecting optical waveguides (ARROWs). Solid-core Y-splitting ARROW waveguides are fabricated orthogonal to the liquid-core section of the chip, creating multiple excitation spots for the analyte. We derive expressions for the SNR increase after signal processing, and analyze its dependence on signal levels and spot number. Very good agreement between theoretical calculations and experimental results is found. SNR enhancements up to 5x104 are demonstrated.

Optimization of Y-splitting antiresonant reflecting optical waveguides-based rib waveguides.

Antiresonant reflecting optical waveguide power splitters, designed for use around the 635-nm wavelength, are characterized for multiple split angles ranging from 0.5 deg to 9 deg. Theoretical expectations and simulations predict lowest transmission losses at this split junction for the lowest angles. This is confirmed by the experimental structures built in SiO2 films on silicon substrates. A fabrication nonideality affects the achievable splitting angle. Design considerations are discussed based on tradeoffs between loss and the required length for a Y-splitter.

Optofluidic wavelength division multiplexing for single-virus detection.

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.