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1.
Proc Natl Acad Sci U S A ; 121(16): e2400203121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38598338

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 Nucleico
2.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33947795

RESUMO

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.


Assuntos
Antígenos Virais/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Diagnóstico Molecular/métodos , SARS-CoV-2/isolamento & purificação , Antígenos Virais/imunologia , Técnicas Biossensoriais , COVID-19/diagnóstico , Fluorescência , Humanos , Vírus da Influenza A/imunologia , Influenza Humana/diagnóstico , Dispositivos Lab-On-A-Chip , Limite de Detecção , Nasofaringe/virologia , Sistemas Automatizados de Assistência Junto ao Leito , SARS-CoV-2/imunologia
3.
Anal Chem ; 93(17): 6739-6745, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33885280

RESUMO

This paper compares static (i.e., temporally unchanging) thermal gradient gas chromatography (GC) to isothermal GC using a stochastic transport model to simulate peak characteristics for the separation of C12-C14 hydrocarbons resulting from variations in injection bandwidth. All comparisons are made using chromatographic conditions that give approximately equal analyte retention times so that the resolution and number of theoretical plates can be clearly compared between simulations. Simulations show that resolution can be significantly improved using a linear thermal gradient along the entire column length. This is mainly achieved by partially compensating for loss in resolution from the increase in mobile phase velocity, which approximates an ideal, basic separation. The slope of the linear thermal gradient required to maximize resolution is a function of the retention parameters, which are specific to each analyte pair; a single static, thermal gradient will not affect all analytes equally. A static, non-linear thermal gradient that creates constant analyte velocities at all column locations provides the largest observed gains in resolution. From the simulations performed in this study, optimized linear thermal gradient conditions are shown to improve the resolution by as much as 8.8% over comparative isothermal conditions, even with a perfect injection (i.e., zero initial bandwidth).

4.
Anal Chem ; 93(34): 11785-11791, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34406737

RESUMO

This paper compares dynamic (i.e., temporally changing) thermal gradient gas chromatography (GC) to temperature-programmed GC using a previously published stochastic transport model to simulate peak characteristics for the separation of C12-C40 hydrocarbons. All comparisons are made using chromatographic conditions that give approximately equal analyte retention times (tR). As shown previously, a static thermal gradient does not improve resolution (Rs) equally for all analytes, which highlights the need for a dynamic thermal gradient. An optimal dynamic thermal gradient should result in constant analyte velocities at any instant in time for those analytes that are actively being separated (i.e., analytes that have low retention factors). The average separation temperature for each analyte is used to determine the thermal gradient profile at different times in the temperature ramp. Because many of the analytes require a similar thermal gradient profile when actively being separated, the thermal gradient profile in this study was held fixed; however, the temperature of the entire thermal gradient was raised over time. From the simulations performed in this study, optimized dynamic thermal gradient conditions are shown to improve Rs by up to 13% over comparative temperature-programmed conditions, even with a perfect injection (i.e., zero injection bandwidth). In the dynamic thermal gradient simulations, all analytes showed improvements in Rs along with slightly shorter tR values compared to simulations for traditional temperature-programmed conditions.


Assuntos
Temperatura , Cromatografia Gasosa
5.
Anal Chem ; 93(4): 2291-2298, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33405883

RESUMO

This article presents a method of simulating molecular transport in capillary gas chromatography (GC) applicable to isothermal, temperature-programmed, and thermal gradient conditions. The approach accounts for parameter differences that can occur across an analyte band including pressure, mobile phase velocity, temperature, and retention factor. The model was validated experimentally using a GC column comprised of microchannels in a stainless-steel plate capable of isothermal, temperature-programmed, and thermal gradient GC separations. The parameters governing retention and dispersion in the transport model were fitted with 12 experimental isothermal separations. The transport model was validated with experimental data for three analytes using four temperature-programmed and three thermal gradient GC separations. The simulated peaks (elution time and dispersion) give reasonable predictions of observed separations. The magnitudes of the maximum error between simulated peak elution time and experiment were 2.6 and 4.2% for temperature-programmed and thermal gradient GC, respectively. The magnitudes of the maximum error between the simulated peak width and experiment were 15.4 and 5.8% for temperature-programmed and thermal gradient GC, respectively. These relatively low errors give confidence that the model reflects the behavior of the transport processes and provides meaningful predictions for GC separations. This transport model allows for an evaluation of analyte separation characteristics of the analyte band at any position along the length of the GC column in addition to peak characteristics at the column exit. The transport model enables investigation of column conditions that influence separation behavior and opens exploration of optimal column design and heating conditions.

6.
J Lightwave Technol ; 39(10): 3330-3340, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34177078

RESUMO

We present a model and simulation for predicting the detected signal of a fluorescence-based optical biosensor built from optofluidic waveguides. Typical applications include flow experiments to determine pathogen concentrations in a biological sample after tagging relevant DNA or RNA sequences. An overview of the biosensor geometry and fabrication processes is presented. The basis for the predictive model is also outlined. The model is then compared to experimental results for three different biosensor designs. The model is shown to have similar signal statistics as physical tests, illustrating utility as a pre-fabrication design tool and as a predictor of detection sensitivity.

7.
Artigo em Inglês | MEDLINE | ID: mdl-33994767

RESUMO

High sensitivity and easy integration with microfabrication techniques has made silicon photonics one of the leading technologies used to build biosensors for diagnostic applications. Here we introduce a new silicon dioxide based optofluidic platform having a planar solid-core (SC) waveguide orthogonally intersecting a liquid-core (LC) waveguide with high refractive index ZnI2 salt solution as core. This enables both more uniform collection of particle fluorescence by the core mode and its propagation to an off-chip detector. This approach results in ultra-high sensitivity performance, demonstrated by achieving 8X enhancement in signal-to-noise ratio, a 45x increase in detection efficiency, and a 100x lower detection limit of 80 aM of fluorescent nanobeads. This represents a key step towards an ultrasensitive biosensor system for analyzing pathogens at clinical concentrations.

8.
Artigo em Inglês | MEDLINE | ID: mdl-33390686

RESUMO

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.

9.
IEEE Photonics Technol Lett ; 33(16): 884-887, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34744399

RESUMO

Optofluidic sensors have enabled single molecule sensing using planar, waveguide dependent multi-spot fluorescence excitation. Here, we demonstrate a new approach to single-particle fluorescence sensing using free-space, top-down illumination of liquid-core antiresonant reflecting optical waveguide (ARROW) devices using two different multi-spot excitation techniques. First, the liquid core ARROW waveguide is excited with a focused beam through a slit pattern milled into an opaque aluminum film, showing comparable performance for single bead fluorescence detection as in-plane, multi-mode interference waveguide based excitation. The second top-down illumination technique images the spot pattern from a Y-splitter SiO2 waveguide chip directly onto the detection device for efficient power utilization and circumventing the need for an opaque cover, producing a further 2.7x improvement in signal-to-noise ratio. The two top-down approaches open up new possibilities for chip-based optical particle sensing with relaxed alignment tolerances.

10.
J Lightwave Technol ; 38(22): 6280-6285, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-33776196

RESUMO

Silica waveguides are often etched by reactive ion etch (RIE) processes. These processes can leave residual topography that can increase optical loss. We investigated the relation between optical loss and various RIE etch. A wet etch step meant to remove microstructures was also considered and compared. Ridge waveguides were fabricated in plasma enhanced chemical vapor deposited films by three different RIE processes, each with a different gas composition, pressure setting, and applied power setting. Half of each set of waveguides were also subjected to a hydrofluoric acid (HF) solution. The waveguides were tested for optical transmission via the cutback method. The transmission vs waveguide length measurements were plotted to fit an exponential curve and the optical loss and measurement uncertainty for each waveguide set was calculated. Clear distinctions in optical loss were found between the different RIE processes. The HF treatment also has an effect, significantly reducing optical loss for two processes and increasing it for the third. Of the tested RIE processes, one can be suggested for silica waveguides. It results in the lowest optical loss and coincidently has the fastest etch rate.

11.
Nanotechnology ; 31(44): 445303, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32679580

RESUMO

We present a method to create robust, nanoscale solid-state membranes using the natural shape of a liquid meniscus as a template. A narrow, open channel is etched into a silicon substrate and then a photoresist polymer is introduced into the channel through spontaneous capillary action. The natural concave meniscus formed by the polymer is then covered by a thin chemical vapor deposited membrane. The polymer is removed by sacrificial etching, leaving behind a suspended membrane. Membranes as large as 20 µm by 9 mm can be fabricated with a thickness as low as 50 nm.

12.
Anal Chem ; 91(1): 792-796, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30525480

RESUMO

This paper reports the first results of a robust, high-performance, stainless-steel microchip gas-chromatography (GC) column that is capable of analyzing complex real-world mixtures as well as operating at very high temperatures. Using a serpentine design, a 10 m column with an approximately semicircular cross-section with a 52 µm hydraulic diameter ( Dh) was produced in a 17 × 6.3 × 0.1 cm rectangular steel chip. The channels were produced using a multilayer-chemical-etch and diffusion-bonding process, and metal nuts were brazed onto the inlet and outlet ports allowing for column interfacing with ferrules and fused silica capillary tubing. After deactivating the metal surface, channels were statically coated with a ≈0.1 µm layer of 5% phenyl-1% vinyl-methylpolysiloxane (SE-54) stationary phase and cross-linked with dicumyl peroxide. By using n-tridecane ( n-C13) as a test analyte with a retention factor ( k) of 5, a total of 44 500 plates (≈4500 plates per meter) was obtained isothermally at 120 °C. The column was thermally stable to at least 350 °C, and rapid temperature programming (35 °C/min) was demonstrated for the boiling-point range from n-C5 to n-C44 (ASTM D2887 simulated-distillation standard). The column was also tested for separation of two complex mixtures: gasoline headspace and kerosene. These initial experiments demonstrate that the planar stainless-steel column with proper interfacing can be a viable alternative platform for portable, robust microchip GC that is capable of high-temperature operation for low-volatility-compound analysis.

13.
Artigo em Inglês | MEDLINE | ID: mdl-30686911

RESUMO

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.

14.
IEEE Photonics Technol Lett ; 30(16): 1487-1490, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30618484

RESUMO

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.

15.
Rapid Commun Mass Spectrom ; 32(23): 2024-2030, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30133876

RESUMO

RATIONALE: Ion trap mass spectrometers are attractive due to their inherent sensitivity and specificity. Miniaturization increases trap portability for in situ mass analysis by relaxing vacuum and voltage requirements but decreases the trapping volume. To overcome signal/resolution loss from miniaturization, double resonance ejection using phase tracking circuitry was investigated. METHODS: Phase tracking circuitry was developed to induce double resonance ejection in a planar linear ion trap using the ß 2/3 hexapole resonance line. RESULTS: Double resonance was observed using phase tracking circuitry. Resolution of 0.5 m/z units and improved signal-to-noise ratio (SNR) compared with AC resonant ejection were achieved. CONCLUSIONS: The phase tracking circuitry proved effective despite deviations from a true phase locked condition. Double resonance ejection is a means to increase signal intensity in a miniaturized planar ion trap.

16.
Rapid Commun Mass Spectrom ; 32(4): 289-294, 2018 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-29131427

RESUMO

RATIONALE: Ion trap mass spectrometers are beneficial due to their intrinsic sensitivity and specificity. Therefore, a portable version for in situ analysis of various compounds is very attractive. Miniaturization of ion traps is paramount for the portability of such mass spectrometers. METHODS: We developed an optimized design for a planar linear ion trap mass spectrometer, consisting of two trapping plates with photolithographically patterned electrodes. Each plate is constructed using a machined glass substrate and standard microfabrication procedures. The plates are attached to a patterned circuit board via wire bonds then positioned approximately 5 mm apart. RESULTS: Trapped ions are detected by ejecting them through tapered slits, which alleviate charge buildup. Mass analysis can be performed through either boundary or resonant ion ejection. Better than unit mass resolution is demonstrated with resonant ejection. CONCLUSIONS: The optimized planar linear ion trap provides good resolution and the potential for further miniaturization. This was accomplished by vigorously testing variables associated with ion trap design including electrical connections, substrate materials, and electrode designs.

17.
IEEE J Quantum Electron ; 54(3)2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29657333

RESUMO

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.

18.
Proc Natl Acad Sci U S A ; 112(42): 12933-7, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26438840

RESUMO

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.


Assuntos
Vírus da Influenza A/isolamento & purificação , Técnicas Analíticas Microfluídicas , Dispositivos Ópticos
19.
IEEE Photonics Technol Lett ; 29(10): 806-809, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29200798

RESUMO

Water absorption was studied in two types of waveguides made from unannealed plasma enhanced chemical vapor deposition (PECVD) SiO2. Standard rib anti-resonant reflecting optical waveguides (ARROWs) were fabricated with thin films of different intrinsic stress and indices of refraction. Buried ARROWs (bARROWs) with low and high refractive index differences between the core and cladding regions were also fabricated from the same types of PECVD films. All waveguides were subjected to a heated, high humidity environment and their optical throughput was tested over time. Due to water absorption in the SiO2 films, the optical throughput of all of the ARROWs decreased with time spent in the wet environment. The ARROWs with the lowest stress SiO2 had the slowest rate of throughput change. High index difference bARROWs showed no decrease in optical throughput after 40 days in the wet environment and are presented as a solution for environmentally stable waveguides made from unannealed PECVD SiO2.

20.
Plant Dis ; 101(5): 738-743, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-30678574

RESUMO

Very little is known about the biology, epidemiology, and best practices to manage Lambertella rot, a newly identified postharvest disease caused by Lambertella corni-maris on apples in the United States. In this study, we investigated the prevalence of L. corni-maris in 92 grower lots throughout Washington State in 2016, evaluated the effect of nutrient availability on L. corni-maris growth, and L. corni-maris sensitivity to pH, osmotic stress, and fungicides in vitro. We assessed pathogen aggressiveness on major apple cultivars and the efficacy of pre- and postharvest fungicides to control L. corni-maris on detached fruit. L. corni-maris was widespread and was found in 40% of the growers lots surveyed at frequencies ranging from 2 to 40% of the total decay. The fungus grew faster on acidic media such as apple juice agar and V8 agar media and was able to grow equally at pH values ranging from 3 to 7. L. corni-maris isolates showed relatively low sensitivity to osmotic stress and grew evenly at 1M KCl. All nine apple cultivars tested were susceptible to L. corni-maris, but the disease severity was significantly higher on some cultivars such as Honeycrisp, Piñata, and Gala. The baseline sensitivity of 100 L. corni-maris isolates was determined based on the effective concentration necessary to inhibit 50% mycelial growth (EC50) for fludioxonil, boscalid, pyraclostrobin + SHAM, pyrimethanil, and thiabendazole. The respective mean EC50 values were 0.07, 0.84, 1.32, 2.45, and 3.68 µg/ml. Fludioxonil and pyrimethanil applied at label rates were the only fungicides able to control L. corni-maris effectively on detached apple fruit. This study is a first step toward understanding the eco-physiological requirements for L. corni-maris to survive and cause decay on apple, assessing potential shifts in fungicide sensitivity, and enhancing Lambertella rot management. We suggest "yellow rot" as the new common name for Lambertella rot.

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