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Recently, studies have revealed that human herpesvirus 4 (HHV-4), also known as the Epstein-Barr virus, might be associated with the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared to SARS-CoV-2 infection alone, patients coinfected with SARS-CoV-2 and HHV-4 had higher risks of fever, inflammation, and even death, thus, confirming that HHV-4/SARS-CoV-2 coinfection in patients could benefit from clinical investigation. Although several intelligent devices can simultaneously discern multiple genes related to SARS-CoV-2, most operate via label-based detection, which restricts them from directly measuring the product. In this study, we developed a device that can replicate and detect SARS-CoV-2 and HHV-4 DNA. This device can conduct a duplex polymerase chain reaction (PCR) in a microfluidic channel and detect replicates in a non-labeled manner through a plasmonic-based sensor. Compared to traditional instruments, this device can reduce the required PCR time by 55% while yielding a similar amount of amplicon. Moreover, our device's limit of detection (LOD) reached 100 fg/mL, while prior non-labeled sensors for SARS-CoV-2 detection were in the range of ng/mL to pg/mL. Furthermore, the device can detect desired genes by extracting cells artificially infected with HHV-4/SARS-CoV-2. We expect that this device will be able to help verify HHV-4/SARS-CoV-2 coinfected patients and assist in the evaluation of practical treatment approaches.
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This paper reports a novel micro/nanostructure co-hot embossing technique. Gold-capped nanostructures were used as localized surface plasmon resonance (SPR) sensors and were integrated into a microfluidic channel. The advantage of the co-hot embossing technique is that the SPR sensors do not need to be aligned with the microfluidic channel while bonding to it. The integrated SPR sensor and microfluidic channel were first characterized, and the sensitivity of the SPR sensor to the refractive index was found using different concentrations of glycerol solutions. The SPR sensor was also used to quantify latent membrane protein (LMP-1) when modifying anti-LMP-1 at the surface of the SPR sensor. Different concentrations of LMP-1 samples were used to build a calibration curve.
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Técnicas Biosensibles , Resonancia por Plasmón de Superficie , Técnicas Biosensibles/métodos , Oro/química , Dispositivos Laboratorio en un Chip , Proteínas de la Membrana , Resonancia por Plasmón de Superficie/métodosRESUMEN
C-Reactive protein (CRP) is an essential biomarker relevant to various disease prognoses. Current biosensors require a significant amount of time for detecting CRP. To address this issue, this work proposes electrokinetic flow-assisted molecule trapping integrated with an impedance biosensor, where a driving signal in terms of a gated sine wave is provided to circularly arranged electrodes which detect proteins. To verify the biosensor's efficacy, protein aggregation on the electrode surface was evaluated through a fluorescence analysis and measurement of the electrochemical impedance spectrum (EIS). The fluorescence analysis with avidin showed that target samples largely accumulated on the electrode surface upon provision of the driving signal. The EIS measurement of CRP accumulation on the electrode surface further confirmed a significant electrokinetic phenomenon at the electrode/electrolyte interface. Even at the low CRP concentration of 10 pg/ml, the proposed device's sensitivity and reliability were as high as 3.92 pg/ml with a signal-to noise ratio (SNR) of ≥3, respectively. In addition, the protein detection time (without considering the preparation time) was minimized to as low as 90 s with the proposed device. This device's advantage is its minimal time consumption, and simple drop-analysis process flow; hence, it was used for monitoring clinical serum samples.
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Técnicas Biosensibles , Proteína C-Reactiva/análisis , Técnicas Electroquímicas , Electrodos , Reproducibilidad de los ResultadosRESUMEN
In this study, microneedle-integrated light sheet microscopy (LSM) was developed for trapping and continuously imaging embryos of Caenorhabditis elegans with subcellular resolution. To reduce aberrations when the light sheet was propagated into the device, a microneedle was fabricated using a transparent, water refractive index-matched polymer. It was proven that when the light sheet emerged from the water-immersed objective and penetrated through the microneedle with a circular surface, even with a non-perpendicular incident angle, fewer aberrations were found. An embryo was injected into and trapped at the tip of the microneedle, which was positioned at the interrogation window of the LSM apparatus with the image plane perpendicular to the light sheet, and this setup was used to sequentially acquire embryo images. By applying the light sheet, higher-resolution, higher-contrast images were obtained. The system also showed low photobleaching and low phototoxicity to embryos of C. elegans. Furthermore, three-dimensional embryo images with a whole field of view of the microneedle could be achieved by stitching together images and reconstructing sequential two-dimensional embryo images.
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Microscopía , Refractometría , Animales , Caenorhabditis elegans , Microscopía/métodos , Fotoblanqueo , AguaRESUMEN
We present the first combination of a microfluidic polymerase chain reaction (PCR) with a gold nanoslit-based surface plasmon resonance (SPR) sensor for detecting the DNA sequence of latent membrane protein 1 (LMP1). The PCR microchannel was produced through a laser scribing technique, and the SPR nanoslit chip was manufactured via hot-embossing nanoimprinting lithography. Afterward, the LMP1 DNA probe was adsorbed onto the SPR chip of the integrated device through electrostatic interactions for further detection. The device can complete the analytical procedure in around 36 min, while the traditional machine requires 105 min to achieve similar signals under the same PCR thermal cycles. The calibration curve with serially diluted LMP1 DNA exhibited the accuracy (R2 > 0.99) and sensitivity (limit of detection: â¼10-11 g/mL) of the device. Moreover, extracted DNA from Epstein-Barr virus (EBV)-positive cells were directly detected through the integrated chip. In brief, this all-in-one chip can amplify gene fragments at the front-end and detect them at the back-end, decreasing the time required for the analysis without compromising accuracy or sensitivity. We believe this label-free, real-time, low-cost device has enormous potential for rapid detection of various viruses, such as EBV and COVID-19.
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Técnicas Biosensibles , COVID-19 , Infecciones por Virus de Epstein-Barr , Oro , Herpesvirus Humano 4/genética , Humanos , Microfluídica , Reacción en Cadena de la Polimerasa , SARS-CoV-2 , Proteínas de la Matriz Viral/genéticaRESUMEN
In this study, a multiplex detection system was proposed by integrating a localized surface plasmon resonance (LSPR) sensing array and parallel microfluidic channels. The LSPR sensing array was fabricated by nanoimprinting and gold sputter on a polycarbonate (PC) substrate. The polydimethylsiloxane (PDMS) microfluidic channels and PC LSPR sensing array were bound together through (3-aminopropyl)triethoxysilane (APTES) surface treatment and oxygen plasma treatment. The resonant spectrum of the LSPR sensing device was obtained by broadband white-light illumination and polarized wavelength measurements with a spectrometer. The sensitivity of the LSPR sensing device was measured using various ratios of glycerol to water solutions with different refractive indices. Multiplex detection was demonstrated using human immunoglobulin G (IgG), IgA, and IgM. The anti-IgG, anti-IgA, and anti-IgM were separately modified in each sensing region. Various concentrations of human IgG, IgA, and IgM were prepared to prove the concept that the parallel sensing device can be used to detect different targets.
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In this study, we developed a portable smartphone-based diffusometry for analyzing the C-reactive protein (CRP) concentration. An optimized fluorescence microscopic add-on system for a smartphone was used to image the 300 nm fluorescent beads. Sequential nanobead images were recorded for a period and the image data were used for fluorescence correlation spectrometric (FCS) analysis. Through the analysis, the nanobeads' diffusion coefficient was obtained. Further, the diffusion coefficients of the anti-CRP-coated nanobeads, which were suspended in the samples with various CRP concentrations, were estimated using smartphone-based diffusometry. After 10 min of reaction, the anti-CRP-coated nanobeads in a higher CRP concentration solution led to a lower diffusion coefficient. Based on the experiments, a linear sensing range of 1~8 µg/mL was found.
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Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Proteína C-Reactiva/química , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Fluorescencia , Pruebas Inmunológicas/instrumentación , Pruebas Inmunológicas/métodos , Nanopartículas/química , Teléfono Inteligente , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodosRESUMEN
Surface plasmon resonance (SPR) nanowire array chips with a microfluidic system are an effective detection method for a rapid test device. This study investigated a capped gold nanowire array and a microfluidic test platform to provide a fundamental understanding of the kinetic binding of SPR nanowires and the surface gold refractive index. The device sensitivity of the SPR nanowire array was 485 nm RIU-1 and the detection limit was 4.1 × 10-5 RIU. Moreover, a kinetic binding analysis also indicated that a peak shift resulted from a specific hybridization of the target molecule with the immobilized probe on the gold nanostructures. The peak shift (red-shift) value of latent membrane protein 1 (LMP1) DNA was 2.21 nm. The results demonstrated that this new method had high sensitivity to detect amplified DNA products without labeling or complex sample treatment. The SPR nanowire chip can detect the PCR products at lower cycle numbers compared to gel electrophoresis due to probe and DNA specificity. Furthermore, the mechanisms of SPR nanowire array fabrication and the detection of the LMP1 gene were studied. The findings can assist in improving the biosensing of DNA-amplified products and in developing rapid detection devices with a small-footprint nanostructured SPR chip.
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ADN Viral/análisis , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Nanocables/química , Resonancia por Plasmón de Superficie/métodos , Proteínas de la Matriz Viral/genética , Secuencia de Bases , Técnicas Biosensibles/métodos , Sondas de ADN/química , Sondas de ADN/genética , ADN Viral/genética , Oro/química , Herpesvirus Humano 4/química , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genética , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentación , Hibridación de Ácido NucleicoRESUMEN
In this study, we developed a new immunosensor that can achieve an ultralow detection limit and high sensitivity. This new device has an electrokinetic trapping (EKT)-based nanofluidic preconcentrator, which was integrated with oscillating bubble valves, to trap concentrated antigen and immunobeads. During the immunoassay process, oscillating bubbles rapidly grew and acted as control valves and to block the microchannel. Thereafter, the trapped preconcentrated antigen plug and antibody-coated nanobeads were preserved in the region between these two valves. Finally, the antigen concentration was quantitatively analyzed by a real-time measurement of Brownian diffusion of the immunobeads. In this work, the test sample used was C-reactive protein (CRP) which is a risk indicator of coronary heart disease and atherosclerosis.
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Proteína C-Reactiva/análisis , Inmunoensayo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Anticuerpos Inmovilizados/química , Difusión , Diseño de Equipo , Humanos , Dispositivos Laboratorio en un Chip , SonidoRESUMEN
An electrokinetic trapping (EKT)-based nanofluidic preconcentration device with the capability of label-free monitoring trapped biomolecules through real-time dual-loop electric current measurement was demonstrated. Universal current-voltage (I-V) curves of EKT-based preconcentration devices, consisting of two microchannels connected by ion-selective channels, are presented for functional validation and optimal operation; universal onset current curves indicating the appearance of the EKT mechanism serve as a confirmation of the concentrating action. The EKT mechanism and the dissimilarity in the current curves related to the volume flow rate (Q), diffusion coefficient (D), and diffusion layer (DL) thickness were explained by a control volume model with a five-stage preconcentration process. Different behaviors of the trapped molecular plug were categorized based on four modes associated with different degrees of electroosmotic instability (EOI). A label-free approach to preconcentrating (bio)molecules and monitoring the multibehavior molecular plug was demonstrated through real-time electric current monitoring, rather than through the use of microscope images.
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Técnicas Electroquímicas/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Nanotecnología/instrumentación , Electricidad , Diseño de EquipoRESUMEN
This paper compares the selectivity and discusses the response mechanisms of various surface-modified, single-walled carbon nanotube (SWCNT)-coated sensor arrays for the detection of volatile organic compounds (VOCs). Two types of sensor platforms, chemiresistor and quartz crystal microbalance (QCM), were used to probe the resistance changes and absorption masses during vapor sensing. Four sensing materials were used in this comparison study: pristine, acidified, esterified, and surfactant (sodium dodecyl sulfate, SDS)-coated SWCNTs. SWCNT-coated QCMs reached the response equilibrium faster than the chemiresistors did, which revealed a delay diffusion behavior at the inter-tube junction. In addition, the calibration lines for QCMs were all linear, but the chemiresistors showed curvature calibration lines which indicated less effectiveness of swelling at high concentrations. While the sorption of vapor molecules caused an increase in the resistance for most SWCNTs due to the swelling, the acidified SWCNTs showed no responses to nonpolar vapors and a negative response to hydrogen bond acceptors. This discovery provided insight into the inter-tube interlocks and conductivity modulation of acidified SWCNTs via a hydrogen bond. The results in this study provide a stepping-stone for further understanding of the mechanisms behind the vapor selectivity of surface-modified SWCNT sensor arrays.
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Fulerenos/química , Gases/análisis , Nanotubos de Carbono/química , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Tensoactivos/química , Compuestos Orgánicos Volátiles/análisis , Ácidos/química , Calibración , Éteres/química , Enlace de HidrógenoRESUMEN
A new technique is developed to measure the nanoparticles' brownian motions by employing microparticle-tracking velocimetry (micro-PTV) in evanescent wave field, which can provide high signal-to-noise ratio images for analyzing nanoparticles' movements. This method enables real-time detection of C-reactive proteins (CRPs) during the rapid interaction between CRPs and anti-CRP-coated nanobeads as CRP concentrations are related to the nanobeads' brownian velocity in the equilibrium state. The smallest observable nanobeads with 185 nm were utilized in this experiment to detect CRP concentrations as low as 0.1 microg/mL even in a high-viscosity solution. Further, the dissociation constant, K(D), can be evaluated based on the experimental results.
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Proteína C-Reactiva/análisis , Nanopartículas/análisis , Reología/métodos , Espectrometría de Fluorescencia , ViscosidadRESUMEN
A novel bio-sensing technique based on the measurement of nanobeads' Brownian motion using a micro-particle tracking velocimetry (micro-PTV) has been successfully developed to detect antigen-antibody interactions. The rapid interaction between antigens (C-reactive proteins, CRPs) and nanobeads with conjugated antibodies (anti-CRPs) has enabled real-time detection of CRPs to be easily carried out. During the binding process of CRPs to nanobeads, the mean value of the beads' diameters increases so that the Brownian velocity decreases with the increase of time. Moreover, higher CRP concentration leads to a lower Brownian velocity of the nanobeads in the equilibrium state. From the results, the limit of detection 0.1microg/ml was observed and could be further improved by using smaller nanobeads. Moreover, based on kinetic analysis, the average dissociation constant K(D)=(6.48+/-1.43)x10(-7) found by this technique for anti-CRP was shown to be in close agreement with the literature values obtained by dual-polarization interferometry (DPI) and indirect competition enzyme-linked immunosorbent assay (ELISA). This simple sensing method can further be used to detect other bio-molecules and viruses.