RESUMEN
Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.
Asunto(s)
Arabidopsis/citología , Arabidopsis/metabolismo , Señalización del Calcio , Calcio/metabolismo , Glicoesfingolípidos/metabolismo , Células Vegetales/metabolismo , Cloruro de Sodio/metabolismo , Arabidopsis/genética , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mutación , Estrés Salino/genética , Estrés Salino/fisiología , Cloruro de Sodio/farmacología , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
The detection of microRNAs (miRNAs) is emerging as a clinically important tool for the non-invasive detection of a wide variety of diseases ranging from cancers and cardiovascular illnesses to infectious diseases. Over the years, miRNA detection schemes have become accessible to clinicians, but they still require sophisticated and bulky laboratory equipment and trained personnel to operate. The exceptional computing ability and ease of use of modern smartphones coupled with fieldable optical detection technologies can provide a useful and portable alternative to these laboratory systems. Herein, we present the development of a smartphone-based device called Krometriks, which is capable of simple and rapid colorimetric detection of microRNA (miRNAs) using a nanoparticle-based assay. The device consists of a smartphone, a 3D printed accessory, and a custom-built dedicated mobile app. We illustrate the utility of Krometriks for the detection of an important miRNA disease biomarker, miR-21, using a nanoplasmonics-based assay developed by our group. We show that Krometriks can detect miRNA down to nanomolar concentrations with detection results comparable to a laboratory-based benchtop spectrophotometer. With slight changes to the accessory design, Krometriks can be made compatible with different types of smartphone models and specifications. Thus, the Krometriks device offers a practical colorimetric platform that has the potential to provide accessible and affordable miRNA diagnostics for point-of-care and field applications in low-resource settings.
Asunto(s)
MicroARNs , Nanopartículas , Biomarcadores , Colorimetría , MicroARNs/genética , Teléfono InteligenteRESUMEN
MicroRNAs (miRNAs) play an important role in the regulation of biological processes and have demonstrated great potential as biomarkers for the early detection of various diseases, including esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE), the premalignant metaplasia associated with EAC. Herein, we demonstrate the direct detection of the esophageal cancer biomarker, miR-21, in RNA extracted from 17 endoscopic tissue biopsies using the nanophotonics technology our group has developed, termed the inverse molecular sentinel (iMS) nanobiosensor, with surface-enhanced Raman scattering (SERS) detection. The potential of this label-free, homogeneous biosensor for cancer diagnosis without the need for target amplification was demonstrated by discriminating esophageal cancer and Barrett's esophagus from normal tissue with notable diagnostic accuracy. This work establishes the potential of the iMS nanobiosensor for cancer diagnostics via miRNA detection in clinical samples without the need for target amplification, validating the potential of this assay as part of a new diagnostic strategy. Combining miRNA diagnostics with the nanophotonics technology will result in a paradigm shift in achieving a general molecular analysis tool that has widespread applicability for cancer research as well as detection of cancer. We anticipate further development of this technique for future use in point-of-care testing as an alternative to histopathological diagnosis as our method provides a quick result following RNA isolation, allowing for timely treatment.
Asunto(s)
Biomarcadores de Tumor/análisis , Técnicas Biosensibles/métodos , ADN/química , Ácidos Nucleicos Inmovilizados/química , Nanopartículas del Metal/química , MicroARNs/análisis , Esófago de Barrett/diagnóstico , Biomarcadores de Tumor/genética , ADN/genética , Diagnóstico Diferencial , Neoplasias Esofágicas/diagnóstico , Oro/química , Humanos , Ácidos Nucleicos Inmovilizados/genética , MicroARNs/genética , Hibridación de Ácido Nucleico , Plata/química , Espectrometría RamanRESUMEN
Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.
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Tecnología de Fibra Óptica , MicroARNs/análisis , Oro/química , Nanopartículas del Metal/química , MicroARNs/genética , Plata/química , Espectrometría Raman , Nicotiana/genéticaRESUMEN
The development of rapid, cost-effective DNA detection methods for molecular diagnostics at the point-of-care (POC) has been receiving increasing interest. This article reviews several DNA detection techniques based on plasmonic-active nanochip platforms developed in our laboratory over the last 5 years, including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). DNA probes were used as the recognition elements, and surface-enhanced Raman scattering (SERS) was used as the signal detection method. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the nanochip's plasmonic-active surface. As the field intensity of the surface plasmon decays exponentially as a function of distance, the distance change in turn affects SERS signal intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized nanochips and measuring the SERS signal after appropriate incubation times. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost-effective. The usefulness of the nanochip platform-based techniques for medical diagnostics was illustrated by the detection of host genetic biomarkers for respiratory viral infection and of the dengue virus gene.
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ADN/análisis , Espectrometría Raman/métodos , Animales , Técnicas Biosensibles/economía , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Sondas de ADN/química , Diseño de Equipo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/economía , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Espectrometría Raman/instrumentaciónRESUMEN
Surface-enhanced Raman scattering (SERS)-active plasmonic nanomaterials have become a promising agent for molecular imaging and multiplex detection. Among the wide variety of plasmonics-active nanoparticles, gold nanostars offer unique plasmon properties that efficiently induce strong SERS signals. Furthermore, nanostars, with their small core size and multiple long thin branches, exhibit high absorption cross sections that are tunable in the near-infrared region of the tissue optical window, rendering them efficient for in vivo spectroscopic detection. This study investigated the use of SERS-encoded gold nanostars for in vivo detection. Ex vivo measurements were performed using human skin grafts to investigate the detection of SERS-encoded nanostars through tissue. We also integrated gold nanostars into a biocompatible scaffold to aid in performing in vivo spectroscopic analyses. In this study, for the first time, we demonstrate in vivo SERS detection of gold nanostars using small animal (rat) as well as large animal (pig) models. The results of this study establish the usefulness and potential of SERS-encoded gold nanostars for future use in long-term in vivo analyte sensing.
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Oro/análisis , Nanoestructuras/análisis , Piel/ultraestructura , Espectrometría Raman/métodos , Animales , Diseño de Equipo , Humanos , Masculino , Modelos Animales , Polihidroxietil Metacrilato/química , Ratas Sprague-Dawley , Trasplante de Piel , Espectrometría Raman/instrumentación , Porcinos , Andamios del Tejido/químicaRESUMEN
Developing a simple and efficient nucleic acid detection technology is essential for clinical diagnostics. Here, we describe a new conceptually simple and selective "turn on" plasmonics-based nanobiosensor, which integrates non-enzymatic DNA strand-displacement hybridization for specific nucleic acid target identification with surface-enhanced Raman scattering (SERS) detection. This SERS nanobiosensor is a target label-free, and rapid nanoparticle-based biosensing system using a homogeneous assay format that offers a simple and efficient tool for nucleic acid diagnostics. Our results showed that the nanobiosensor provided a limit of detection of ~0.1nM (200amol) in the current bioassay system, and exhibited high specificity for single nucleotide mismatch discrimination. FROM THE CLINICAL EDITOR: Surface-enhanced Raman scattering (SERS) is a sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement means that the technique may even detect single molecules. In this article, the authors describe a simple and efficient nucleic acid detection technology using SERS, with "OFF-to-ON" signal switch upon nucleic acid target identification and capture, which provides high sensitivity and specificity for single nucleotide mismatch discrimination. This new technology will be most welcomed in clinical diagnostics.
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ADN/análisis , Resonancia por Plasmón de Superficie/métodos , Espectrometría Raman/métodosRESUMEN
A novel DNA bioassay-on-chip using surface-enhanced Raman scattering (SERS) on a bimetallic nanowave chip is presented. In this bioassay, SERS signals were measured after a single reaction on the chip's surface without any washing step, making it simple-to-use and reducing the reagent cost. Using the technique, specific oligonucleotide sequences of the dengue virus 4 were detected.
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Bioensayo , ADN Viral/análisis , Virus del Dengue/genética , Dengue/diagnóstico , Dispositivos Laboratorio en un Chip , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de RastreoRESUMEN
Developing techniques for multiplex detection of disease biomarkers is important for clinical diagnosis. In this work, we have demonstrated for the first time the feasibility of multiplex detection of genetic disease biomarkers using the surface-enhanced Raman scattering (SERS)-based molecular sentinel-on-chip (MSC) diagnostic technology. The molecular sentinel (MS) sensing mechanism is based upon the decrease of SERS intensity when Raman labels tagged at 3'-ends of MS nanoprobes are physically displaced from the nanowave chip's surface upon DNA hybridization. The use of bimetallic layer (silver and gold) for the nanowave fabrication was investigated. SERS measurements were performed immediately following a single hybridization reaction between the target single-stranded DNA sequences and the complementary MS nanoprobes immobilized on the nanowave chip without requiring target labeling (i.e., label-free), secondary hybridization, or post-hybridization washing, thus shortening the assay time and reducing cost. Two nucleic acid transcripts, interferon alpha-inducible protein 27 and interferon-induced protein 44-like, are used as model systems for the multiplex detection concept demonstration. These two genes are well known for their critical role in host immune response to viral infection and can be used as molecular signature for viral infection diagnosis. The results indicate the potential of the MSC technology for nucleic acid biomarker multiplex detection.
Asunto(s)
Bioensayo , Biomarcadores/análisis , Biomarcadores/química , Hibridación de Ácido Nucleico , Espectrometría Raman , Antígenos/química , Técnicas Biosensibles , Proteínas del Citoesqueleto/química , ADN/química , ADN de Cadena Simple/química , Oro/química , Humanos , Sistema Inmunológico , Espectrometría de Masas , Proteínas de la Membrana/química , Microscopía Electrónica de Rastreo , Nanotecnología , Oligonucleótidos/química , Plata/química , Factores de TiempoRESUMEN
The intracellular developmental processes in plants, particularly concerning lignin polymer formation and biomass production are regulated by microRNAs (miRNAs). MiRNAs including miR397b are important for developing efficient and cost-effective biofuels. However, traditional methods of monitoring miRNA expression, like PCR, are time-consuming, require sample extraction, and lack spatial and temporal resolution, especially in real-world conditions. We present a novel approach using plasmonics nanosensing to monitor miRNA activity within living plant cells without sample extraction. Plasmonic biosensors using surface-enhanced Raman scattering (SERS) detection offer high sensitivity and precise molecular information. We used the Inverse Molecular Sentinel (iMS) biosensor on unique silver-coated gold nanorods (AuNR@Ag) with a high-aspect ratio to penetrate plant cell walls for detecting miR397b within intact living plant cells. MiR397b overexpression has shown promise in reducing lignin content. Thus, monitoring miR397b is essential for cost-effective biofuel generation. This study demonstrates the infiltration of nanorod iMS biosensors and detection of non-native miRNA 397b within plant cells for the first time. The investigation successfully demonstrates the localization of nanorod iMS biosensors through TEM and XRF-based elemental mapping for miRNA detection within plant cells of Nicotiana benthamiana. The study integrates shifted-excitation Raman difference spectroscopy (SERDS) to decrease background interference and enhance target signal extraction. In vivo SERDS testing confirms the dynamic detection of miR397b in Arabidopsis thaliana leaves after infiltration with iMS nanorods and miR397b target. This proof-of-concept study is an important stepping stone towards spatially resolved, intracellular miRNA mapping to monitor biomarkers and biological pathways for developing efficient renewable biofuel sources.
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Técnicas Biosensibles , Oro , MicroARNs , Nanotubos , Espectrometría Raman , Nanotubos/química , Técnicas Biosensibles/métodos , MicroARNs/genética , MicroARNs/análisis , Oro/química , Espectrometría Raman/métodos , Nicotiana/genética , Nicotiana/química , Plata/química , Biomarcadores , Lignina/químicaRESUMEN
Efficient isolation and analysis of exosomal biomarkers hold transformative potential in biomedical applications. However, current methods are prone to contamination and require costly consumables, expensive equipment, and skilled personnel. Here, we introduce an innovative spaceship-like disc that allows Acoustic Separation and Concentration of Exosomes and Nucleotide Detection: ASCENDx. We created ASCENDx to use acoustically driven disc rotation on a spinning droplet to generate swift separation and concentration of exosomes from patient plasma samples. Integrated plasmonic nanostars on the ASCENDx disc enable label-free detection of enriched exosomes via surface-enhanced Raman scattering. Direct detection of circulating exosomal microRNA biomarkers from patient plasma samples by the ASCENDx platform facilitated a diagnostic assay for colorectal cancer with 95.8% sensitivity and 100% specificity. ASCENDx overcomes existing limitations in exosome-based molecular diagnostics and holds a powerful position for future biomedical research, precision medicine, and point-of-care medical diagnostics.
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Exosomas , Nucleótidos , Humanos , Biomarcadores , Medicina de Precisión , Espectrometría RamanRESUMEN
Development of a rapid, cost-effective, label-free biosensor for DNA detection is important for many applications in clinical diagnosis, homeland defense, and environment monitoring. A unique label-free DNA biosensor based on Molecular Sentinel (MS) immobilized on a plasmonic 'Nanowave' chip, which is also referred to as a metal film over nanosphere (MFON), is presented. Its sensing mechanism is based upon the decrease of the surface-enhanced Raman scattering (SERS) intensity when Raman label tagged at one end of MS is physically separated from the MFON's surface upon DNA hybridization. This method is label-free as the target does not have to be labeled. The MFON fabrication is relatively simple and low-cost with high reproducibility based on depositing a thin shell of gold over close-packed arrays of nanospheres. The sensing process involves a single hybridization step between the DNA target sequences and the complementary MS probes on the Nanowave chip without requiring secondary hybridization or posthybridization washing, thus resulting in rapid assay time and low reagent usage. The usefulness and potential application of the biosensor for medical diagnostics is demonstrated by detecting the human radical S-adenosyl methionine domain containing 2 (RSAD2) gene, a common inflammation biomarker.
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Técnicas Biosensibles/métodos , ADN/química , Nanotecnología/métodos , Proteínas/análisis , Espectrometría Raman/métodos , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Propiedades de SuperficieRESUMEN
Recent advances in integrating nanotechnology and optical microscopy offer great potential in intracellular applications with improved molecular information and higher resolution. Continuous efforts in designing nanoparticles with strong and tunable plasmon resonance have led to new developments in biosensing and bioimaging, using surface-enhanced Raman scattering and two-photon photoluminescence. We provide an overview of the nanoprobe design updates, such as controlling the nanoparticle shape for optimal plasmon peak position; optical sensing and imaging strategies for intracellular nanoparticle detection; and addressing practical challenges in cellular applications of nanoprobes, including the use of targeting agents and control of nanoparticle aggregation.
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Células/química , Nanopartículas del Metal/química , Sondas Moleculares/química , Nanotecnología/instrumentación , Resonancia por Plasmón de Superficie/instrumentación , Animales , Humanos , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodos , Nanotecnología/métodos , Resonancia por Plasmón de Superficie/métodosRESUMEN
The development of DNA detection techniques on large-area plasmonics-active platforms is critical for many medical applications such as high-throughput screening, medical diagnosis and systems biology research. Here, we report for the first time a unique "molecular sentinel-on-chip" (MSC) technology for surface-enhanced Raman scattering (SERS)-based DNA detection. This unique approach allows label-free detection of DNA molecules on chips developed on a wafer scale using large area nanofabrication methodologies. To develop plasmonics-active biosensing platforms in a repeatable and reproducible manner, we employed a combination of deep UV lithography, atomic layer deposition, and metal deposition to fabricate triangular-shaped nanowire (TSNW) arrays having controlled sub-10 nm gap nanostructures over an entire 6 inch wafer. The detection of a DNA sequence of the Ki-67 gene, a critical breast cancer biomarker, on the TSNW substrate illustrates the usefulness and potential of the MSC technology as a novel SERS-based DNA detection method.
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Técnicas Biosensibles , ADN/análisis , Espectrometría Raman , Biomarcadores/análisis , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Antígeno Ki-67/genética , Nanocables/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/metabolismo , Silicio/químicaRESUMEN
Recent advances in molecular technologies have provided various assay strategies for monitoring biomarkers, such as miRNAs for early detection of various diseases and cancers. However, there is still an urgent unmet need to develop practical and accurate miRNA analytical tools that could facilitate the incorporation of miRNA biomarkers into clinical practice and management. In this study, we demonstrate the feasibility of using a cascade amplification method, referred to as the "Cascade Amplification by Recycling Trigger Probe" (CARTP) strategy, to improve the detection sensitivity of the inverse Molecular Sentinel (iMS) nanobiosensor. The iMS nanobiosensor developed in our laboratory is a unique homogeneous multiplex bioassay technique based on surface-enhanced Raman scattering (SERS) detection, and was used to successfully detect miRNAs from clinical samples. The CARTP strategy based on the toehold-mediated strand displacement reaction is triggered by a linear DNA strand, called the "Recycling Trigger Probe" (RTP) strand, to amplify the iMS SERS signal. Herein, by using the CARTP strategy, we show a significantly improved detection sensitivity with the limit of detection (LOD) of 45 fM, which is 100-fold more sensitive than the non-amplified iMS assay used in our previous report. We envision that the further development and optimization of this strategy ultimately will allow multiplexed detection of miRNA biomarkers with ultra-high sensitivity for clinical translation and application.
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Bioensayo , MicroARNs , Laboratorios , Límite de Detección , ReciclajeRESUMEN
There is a critical need for sensitive and rapid detection technologies utilizing molecular biotargets such as microRNAs (miRNAs), which regulate gene expression and are a promising class of diagnostic biomarkers for disease detection. Here, we present the development and fabrication of a highly reproducible and robust plasmonic bimetallic nanostar biosensing platform to detect miRNA targets using surfaced-enhanced Raman scattering (SERS)-based gene probes called the inverse Molecular Sentinel (iMS). We investigated and optimized the integration of iMS gene probes onto this SERS substrate, achieving ultra-sensitive detection with limits of detection of 6.8 and 16.7 zmol within the sensing region for two miRNA sequences of interest. Finally, we demonstrated the biomedical usefulness of this nanobiosensor platform with the multiplexed detection of upregulated miRNA targets, miR21 and miR221, from colorectal cancer patient plasma. The resulting SERS data are in excellent agreement with PCR data obtained from patient samples and can distinguish between healthy and cancerous patient samples. These results underline the potential of the iMS-integrated substrate nanobiosensing platform for rapid and sensitive diagnostics of cancer biomarkers for point-of-care applications.
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Técnicas Biosensibles , Neoplasias Colorrectales , Nanopartículas del Metal , MicroARNs , Humanos , Técnicas Biosensibles/métodos , MicroARNs/análisis , Biomarcadores de Tumor/genética , Nanopartículas del Metal/química , Espectrometría Raman/métodos , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Oro/química , Límite de DetecciónRESUMEN
A label-free approach using plasmonic coupling interference (PCI) nanoprobes for nucleic acid detection using surface-enhanced Raman scattering (SERS) is described. To induce a strong plasmonic coupling effect, a nanonetwork of silver nanoparticles with the Raman label located between adjacent nanoparticles is assembled by Raman-labeled DNA-locked nucleic acid (LNA) duplexes. The PCI method then utilizes specific nucleic acid sequences of interest as competitor elements for the Raman-labeled DNA strands to interfere the formation of nanonetworks in a competitive binding process. As a result, the plasmonic coupling effect induced through the formation of the nanonetworks is significantly diminished, resulting in a reduced SERS signal. The potential of the PCI technique for biomedical applications is illustrated by detecting single-nucleotide polymorphism (SNP) and microRNA sequences involved in breast cancers. The results of this study could lead to the development of nucleic acid diagnostic tools for biomedical diagnostics and biosensing applications using SERS detection.
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Nanopartículas del Metal/química , Ácidos Nucleicos/análisis , Espectrometría Raman/métodos , ADN/química , MicroARNs/química , Polimorfismo de Nucleótido Simple , Plata/químicaRESUMEN
A critical aspect for use of nanoprobes in biomedical research and clinical applications involves fundamental spatial and temporal characterization of their uptake and distribution in cells. Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate nanoprobes in single cells using surface-enhanced Raman scattering detection. To study the efficiency of cellular uptake, silver nanoparticles functionalized with three different positive-, negative-, and neutrally charged Raman labels were co-incubated with cell cultures and internalized via normal cellular processes. The surface charge on the nanoparticles was observed to modulate uptake efficiency, demonstrating a dual function of the surface modifications as tracking labels and as modulators of cell uptake. These results indicate that the functionalized nanoparticle construct has potential for sensing and delivery in single living cells and that use of surface-enhanced Raman scattering for tracking and detection is a practical and advantageous alternative to traditional fluorescence methods. FROM THE CLINICAL EDITOR: Cell labeling and tracking methods are commonly required in biomedical research. This paper presents specific functionalized nanoparticle constructs with potential for sensing and delivery in single living cells. The use of surface-enhanced Raman scattering enables tracking and detection of these cells as a practical alternative to traditional fluorescence methods.
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Nanopartículas/química , Plata/química , Espectrometría Raman/métodos , Animales , Línea Celular , RatonesRESUMEN
We have demonstrated for the first time the feasibility of multiplex detection using the surface-enhanced Raman scattering-based molecular sentinel (MS) technology in a homogeneous solution. Two MS nanoprobes tagged with different Raman labels were used to detect the presence of the erbB-2 and ki-67 breast cancer biomarkers. The multiplexing capability of the MS technique was demonstrated by mixing the two MS nanoprobes and tested in the presence of single or multiple DNA targets.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Antígeno Ki-67/genética , Hibridación de Ácido Nucleico/métodos , Receptor ErbB-2/genética , Biomarcadores de Tumor/genética , Técnicas de Laboratorio Clínico/métodos , Humanos , Nanotecnología/métodos , Espectrometría RamanRESUMEN
MicroRNAs (miRNAs), small noncoding endogenous RNA molecules, are emerging as promising biomarkers for early detection of various diseases and cancers. Practical screening tools and strategies to detect these small molecules are urgently needed to facilitate the translation of miRNA biomarkers into clinical practice. In this study, a label-free biosensing technique based on surface-enhanced Raman scattering (SERS), referred to as plasmonic coupling interference (PCI), was applied for the multiplex detection of miRNA biomarkers. The sensing mechanism of the PCI technique relies on the formation of a nanonetwork consisting of nanoparticles with Raman labels located between adjacent nanoparticles that are interconnected by DNA duplexes. Because of the plasmonic coupling effect of adjacent nanoparticles in the nanonetwork, the Raman labels exhibit intense SERS signals. Such effect can be modulated by the addition of miRNA targets of interest that act as inhibitors to interfere with the formation of this nanonetwork, resulting in a diminished SERS signal. In this study, the PCI technique is theoretically analyzed, and the multiplex capability for detection of multiple miRNA cancer biomarkers is demonstrated, establishing the great potential of PCI nanoprobes as a useful diagnostic tool for medical applications.