RESUMO
Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.
Assuntos
Nanoporos , Infecção por Zika virus , Zika virus , Animais , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Primatas/genética , Zika virus/genética , Sensibilidade e Especificidade , Técnicas de Amplificação de Ácido NucleicoRESUMO
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.
Assuntos
Coloides/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Nanopartículas/análise , Nanopartículas/química , Pinças Ópticas , Ressonância de Plasmônio de Superfície/instrumentação , Coloides/química , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Bacterial nanowires are extracellular appendages that have been suggested as pathways for electron transport in phylogenetically diverse microorganisms, including dissimilatory metal-reducing bacteria and photosynthetic cyanobacteria. However, there has been no evidence presented to demonstrate electron transport along the length of bacterial nanowires. Here we report electron transport measurements along individually addressed bacterial nanowires derived from electron-acceptor-limited cultures of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. Transport along the bacterial nanowires was independently evaluated by two techniques: (i) nanofabricated electrodes patterned on top of individual nanowires, and (ii) conducting probe atomic force microscopy at various points along a single nanowire bridging a metallic electrode and the conductive atomic force microscopy tip. The S. oneidensis MR-1 nanowires were found to be electrically conductive along micrometer-length scales with electron transport rates up to 10(9)/s at 100 mV of applied bias and a measured resistivity on the order of 1 Ω·cm. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA produce appendages that are morphologically consistent with bacterial nanowires, but were found to be nonconductive. The measurements reported here allow for bacterial nanowires to serve as a viable microbial strategy for extracellular electron transport.
Assuntos
Eletricidade , Shewanella/fisiologia , Eletrodos , Microscopia de Força Atômica , NanofiosRESUMO
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.
Assuntos
Técnicas Biossensoriais , COVID-19 , Nanoporos , Teste para COVID-19 , Humanos , Pinças Ópticas , Pandemias , RNA Viral/genética , SARS-CoV-2RESUMO
Solid-state nanopores can be fabricated in a variety of ways and form the basis for label-free sensing of single nanoparticles: as individual nanoparticles traverse the nanopore, they alter the ionic current across it in a characteristic way. Typically, nanopores are described by the diameter of their limiting aperture, and less attention has been paid to other, fabrication-dependent parameters. Here, we report a comprehensive analysis of the properties and sensing performance of three types of nanopore with identical 50 nm aperture, but fabricated using three different techniques: direct ion beam milling, ion beam sculpting, and electron beam sculpting. The nanopores differ substantially in physical shape and chemical composition as identified by ion-beam assisted cross-sectioning and energy dispersive X-ray spectroscopy. Concomitant differences in electrical sensing of single 30 nm beads, such as variations in blockade depth, duration, and electric field dependence, are observed and modeled using hydrodynamic simulations. The excellent agreement between experiment and physical modeling shows that the physical properties (shape) and not the chemical surface composition determine the sensing performance of a solid-state nanopore in the absence of deliberate surface modification. Consequently, nanoparticle sensing performance can be accurately predicted once the full three-dimensional structure of the nanopore is known.
RESUMO
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.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Dispositivos Ópticos , Integração de Sistemas , Animais , DNA/análise , Dimetilpolisiloxanos/química , Microesferas , Fibras Ópticas , Silício/químicaRESUMO
OBJECTIVE: Bacterial biofilms play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this preliminary study was to test the hypothesis that the extracellular filaments observed in biofilms associated with BRONJ contain electrically conductive nanowires. STUDY DESIGN: Bone samples of patients affected by BRONJ were evaluated for conductive nanowires by scanning electron microscopy (SEM) and conductive probe atomic force microscopy (CP-AFM). We created nanofabricated electrodes to measure electrical transport along putative nanowires. RESULTS: SEM revealed large-scale multispecies biofilms containing numerous filamentous structures throughout necrotic bone. CP-AFM analysis revealed that these structures were electrically conductive nanowires with resistivities on the order of 20 Ω·cm. Nanofabricated electrodes spaced along the nanowires confirmed their ability to transfer electrons over micron-scale lengths. CONCLUSIONS: Electrically conductive bacterial nanowires to date have been described only in environmental isolates. This study shows for the first time that these nanowires can also be found in clinically relevant biofilm-mediated diseases, such as BRONJ, and may represent an important target for therapy.