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.

Enhanced Detection of Single Viruses On-Chip via Hydrodynamic Focusing.

Planar optofluidics provide a powerful tool for facilitating chip-scale light-matter interactions. Silicon-based liquid core waveguides have been shown to offer single molecule sensitivity for efficient detection of bioparticles. Recently, a PDMS based planar optofluidic platform was introduced that opens the way to rapid development and prototyping of unique structures, taking advantage of the positive attributes of silicon dioxide-based optofluidics and PDMS based microfluidics. Here, hydrodynamic focusing is integrated into a PDMS based optofluidic chip to enhance the detection of single H1N1 viruses on-chip. Chip-plane focusing is provided by a system of microfluidic channels to force the particles towards a region of high optical collection efficiency. Focusing is demonstrated and enhanced detection is quantified using fluorescent polystyrene beads where the coefficient of variation is found to decrease by a factor of 4 with the addition of hydrodynamic focusing. The mean signal amplitude of fluorescently tagged single H1N1 viruses is found to increase with the addition of focusing by a factor of 1.64.

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.

Integration of sample preparation and analysis into an optofluidic chip for multi-target disease detection.

Detection of molecular biomarkers with high specificity and sensitivity from biological samples requires both sophisticated sample preparation and subsequent analysis. These tasks are often carried out on separate platforms which increases required sample volumes and the risk of errors, sample loss, and contamination. Here, we present an optofluidic platform which combines an optical detection section with single nucleic acid strand sensitivity, and a sample processing unit capable of on-chip, specific extraction and labeling of nucleic acid and protein targets in complex biological matrices. First, on-chip labeling and detection of individual lambda DNA molecules down to concentrations of 8 fM is demonstrated. Subsequently, we demonstrate the simultaneous capture, fluorescence tagging and detection of both Zika specific nucleic acid and NS-1 protein targets in both buffer and human serum. We show that the dual DNA and protein assay allows for successful differentiation and diagnosis of Zika against cross-reacting species like dengue.

Multi-channel velocity multiplexing of single virus detection on an optofluidic chip.

Liquid-core waveguide-based optofluidic devices have proven to be valuable tools for analysis of biological samples in fluid. They have enabled single bioparticle sensitivity while maintaining in-plane detection via light-induced fluorescence. The incorporation of multi-spot excitation with multimode interference (MMI) waveguides has enabled spatially and spectrally multiplexed detection of single viruses on an oxide-based optofluidic platform. Here, we introduce a new way of MMI-based multiplexing where multiple analysis channels are placed within a single multi-spot pattern. This stacked channel design enables both velocity and spectral multiplexing of single particles. The principle is demonstrated with differentiated detection of single H3N2 and H1N1 viruses on a polydimethylsiloxane platform.

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.

Flexible optofluidic waveguide platform with multi-dimensional reconfigurability.

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.

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.

Improved environmental stability for plasma enhanced chemical vapor deposition SiO2 waveguides using buried channel designs.

Ridge and buried channel waveguides (BCWs) made using plasma-enhanced chemical vapor deposition SiO2 were fabricated and tested after being subjected to long 85°C water baths. The water bath was used to investigate the effects of any water absorption in the ridge and BCWs. Optical mode spreading and power throughput were measured over a period of three weeks. The ridge waveguides quickly absorbed water within the critical guiding portion of the waveguide. This caused a nonuniformity in the refractive index profile, leading to poor modal confinement after only seven days. The BCWs possessed a low index top cladding layer of SiO2, which caused an increase in the longevity of the waveguides, and after 21 days, the BCW samples still maintained ~20% throughput, much higher than the ridge waveguides, which had a throughput under 5%.

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.

Electro-optical detection of single λ-DNA.

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.

Correlated electrical and optical analysis of single nanoparticles and biomolecules on a nanopore-gated optofluidic chip.

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.

Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431.

BACKGROUND: Paclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer. RESULTS: The ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely. CONCLUSIONS: Our findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.

Hybrid optofluidic integration.

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.

Dual-core optofluidic chip for independent particle detection and tunable spectral filtering.

We present the first integration of fluidically tunable filters with a separate particle detection channel on a single planar, optofluidic chip. Two optically connected, but fluidically isolated liquid-core antiresonant reflecting optical waveguide (ARROW) segments serve as analyte and spectral filter sections, respectively. Ultrasensitive detection of fluorescent nanobeads with high signal-to-noise ratio provided by a fluidically tuned excitation notch filter is demonstrated. In addition, reconfigurable filter response is demonstrated using both core index tuning and bulk liquid tuning. Notch filters with 43 dB rejection ratio and a record 90 nm tuning range are implemented by using different mixtures of ethylene glycol and water in the filter section. Moreover, absorber dyes and liquids with pH-dependent transmission in the filter channel provide additional spectral control independent of the waveguide response. Using both core index and pH control, independent filter tuning at multiple wavelengths is demonstrated for the first time. This extensive on-chip control over spectral filtering as one of the fundamental components of optical particle detection techniques offers significant advantages in terms of compactness, cost, and simplicity, and opens new opportunities for waveguide-based optofluidic analysis systems.