MicroRNAs (miRNAs) are an emerging biotarget for clinical applications such as non-invasive detection of diseases such as cancer. However, current methods of miRNA quantification require tedious processing and amplification with highly trained specialists. Herein we show a simple and rapid colorimetric smartphone system that takes advantage of plasmonic nanoparticle-based assays to easily quantify miR-21 biomarker as a representative target for cancer disease detection. To achieve this, we use a unique assay known as Plasmonic Coupling Interference (PCI) to optically quantify the amount of biomarkers present. In addition, we utilize a specialized colorimetric processing method paired with accessible and cheap 3D printed parts to obviate the need for specialists to analyze and interpret the data. Our smart phone sensing system offers a practical miRNA diagnostic platform for the point-of-care applications as an alternative to more expensive lab-based methods.
MicroRNAs (miRNAs) have increasingly become an important biomarker target for applications ranging from clinical diagnostics to biofuel production monitoring. However, the current state of the art for the detection of such markers requires tedious processing and amplification techniques such as polymerase chain reaction (PCR). In an effort to create a relatively simple biosensing platform, we have developed a combined plasmonic biosensing method based on a Surface-Enhanced Raman Spectroscopy (SERS) platform called the inverse Molecular Sentinel (iMS) to directly detect in vivo miRNA such as miR858a. With Shifted Excitation Raman Difference Spectroscopy (SERDS), we can remotely detect these targets in the field in the presence of interfering background signal. The application of such technology can pave the way not just for biofuel monitoring but early and non-invasive disease detection and diagnostics.
We present the integration of a miRNA sensing probe technology with a bimetallic nanostar-based surfaced-enhanced Raman scattering (SERS) substrate for disease detection. Circulating microRNAs (miRNA) are being investigated as promising diagnostic biomarkers for many cancers, including colorectal cancer (CRC). The inverse molecular sentinel technology was used for amplification-free multiplexed detection of miRNA targets using SERS. The integration of these technologies lays the foundation for point of care device design, capable of miRNA profiling without the need for traditional sample preparation of nucleotide amplification techniques.
Plant biotechnology and biofuel research is critical in addressing increasing global demands for energy. Further understanding of biomass producing associated metabolic pathways in plants can be used to exploit and increase the production of biomass for energy purposes. In vivo detection of biomarkers associated with plant growth for bioenergy has proved to be limited due to complex sample preparation required by traditional methods. In addition, genetic transformation and biomolecule monitoring inside plant cells is regulated by diameter and size exclusion limits of the plant cell wall (5 - 20 nm). Currently limited methods exist for enabling direct entry into plant cells. Moreover, these methods, such as biolistic particle delivery and electroporation use mechanical force that causes damages to the plant tissue. Nanoparticles could serve as promising platforms for probes to characterize intercellular and intracellular plant biomarkers and pathways. Bi-metallic nanostars are a plasmonics-active nanoplatform capable of high surface-enhanced Raman scattering (SERS) which can enter plant cells and have the future potential for nucleic acid sensing. Imaging technologies such as SERS mapping, confocal imaging, X-ray fluorescence imaging, multi-photon imaging, and transmission electron microscopy have been utilized to determine the compartmentalization and location of the SERS iMS biosensors inside Arabidopsis plants.
Surface-enhanced Raman scattering (SERS) is emerging as an alternative non-invasive detection method in many applications. We recently show the use of SERS nanoprobes to detect tumors in vivo in mice, as well as the use of SERS sensors in vivo in plants for the detection of mIRNA. In spite of these advancements, the translation of SERS to real-world settings has been limited due to issues with observing Raman signal over complex background. For example, it remains challenging to observe SERS under sunlight or under strong illumination (e.g., operating room), using a conventional Raman setup. To this end, we combined a Raman setup with a newly developed dual-wavelength laser to perform shifted-excitation Raman difference spectroscopy (SERDS). Using SERDS, we demonstrate that the use of SERS sensors to detect miRNA in live plants inside a growth chamber, under full illumination. Additionally, we show that SERDS can be used to accurately identify tumors in mice, under ambient light. In both these applications, we demonstrate that the combination of SERS with SERDS improves the sensitivity and accuracy. This work will aid the translation of Raman and SERS to real-world settings.
Our group has integrated surface-enhanced Raman scattering (SERS) silver coated gold nanostars on an optical fiber. Fiber-based sensors are an in-situ technology that can simultaneously bring the sensor and light to the sample without disturbing the environment. This technology is a multi-use method that does not require complex sample preparation. Fiber sensors or optrodes, enable the detection of analytes in samples that are difficult to access. Additionally, optrodes allow for specific detection while evading background signals from non-target regions. The fiber-optrode was used to detect miRNA and illegal food additives.
Further understanding of biomass producing associated metabolic pathways in plants can be used to increase the production of biomass. In vivo detection of these markers has proved to be limited due to complex sample preparation required by traditional methods. Recently the Vo-Dinh group has designed a platform to detect nucleic acid targets in biological systems called inverse molecular sentinels which utilize surface-enhanced Raman scattering. These multimodal probes were shown to detect and image key microRNA within whole plants in vivo. This work lays the foundation for detecting and imaging biological markers in plants with enhanced spatial and temporal resolution.
Development of simple techniques for detection of nucleic acids is essential for clinical diagnostics. MicroRNA (miRNA) have been identified as potential biomarkers in early detection of various diseases, including esophageal adenocarcinoma (EAC) and Barrett’s esophagus (BE). However, these small molecules have not been adopted into clinical practice because of challenging aspects in the lab. The technology described herein is a label-free nanoprobe referred to as “inverse molecular sentinel” (iMS) for gastrointestinal cancer diagnosis using surface-enhanced Raman scattering (SERS) detection. The results of this study provide evidence supporting the robust iMS method for miRNA sensing without the need for PCR.
Molecular analysis has revolutionized many applications, including bio-safety, bio-engineering and biofuel research; however, there are limited practical tools for in situ detection during field work. New technology is needed to translate molecular advances from laboratory settings into the practical realm. The unique characteristics of plasmonic nanosensors have made them ideal candidates for field-ready sensing applications. Herein, we discuss the development of a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of miRNA targets, which are detected 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 to detect target nucleic acids using the surface-enhanced Raman scattering sensing mechanism of nanoprobes referred to as inverse molecular sentinels. The fiber sensors were tested in extracts from leaves of plants that were induced to have different miRNA expression levels. The results indicate that the fiber sensors developed have the potential to be a powerful tool for field analysis.
Gene expression monitoring within whole plants is critical for many applications ranging from plant biology to biofuel development. Herein, we report a unique multimodal method for in vivo imaging and biosensing of nucleic acid biotargets, specifically microRNA, within whole plant leaves by integrating three complementary techniques: surface-enhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The method described utilizes plasmonic nanostar-based inverse molecular sentinel (iMS) nanoprobes, which not only provide large Raman signal enhancement upon target binding, but also allow for localization and quantification by XRF and plasmonics-enhanced TPL. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants.
The ability to monitor gene expression within living plants is of importance in many applications ranging from plant biology research to biofuel development; however, no method currently exists without requiring sample extraction. Herein, we report a multimodal imaging method based on plasmonic nanoprobes for in vivo imaging and biosensing of microRNA biotargets within whole plant leaves. This method integrates three different but complementary techniques: surfaceenhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The multimodal method utilizes plasmonic nanostars, which not only provide large Raman signal enhancement, but also allow for localization and quantification by XRF and plasmonics-enhanced TPL, owing to gold content and high two-photon luminescence cross-sections. For the sensing mechanism, inverse molecular sentinel (iMS) nanoprobes are used for SERS bioimaging of microRNA within Arabidopsis thaliana leaves to provide a dynamic SERS map of detected microRNA targets while also quantifying nanoprobe concentrations using XRF and TPL. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants, a tool that will allow the study of these biotargets with previously unmet spatial and temporal resolution.
Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo molecular detection remains very challenging. Here, we demonstrated the proof‐of‐principle of in vivo detection of nucleic acid targets using a promising type of surface‐enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo “smart tattoo” nanosensor used in this study employs the “inverse molecular sentinel” (iMS) detection scheme, which is a label-free homogeneous biosensing system based on a non-enzymatic DNA strand-displacement process and conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. In this study, plasmonics‐active nanostar was utilized as an efficient in vivo SERS sensing platform due to their tunable absorption bands in the near infrared region of the “tissue optical window. The results of this study illustrate the usefulness of SERS iMS nanosensors as an implantable skin‐based in vivo biosensing platform, providing a foundation for developments in continuous health status sensing, disease biomarker monitoring, and other clinical translation applications.
The knowledge over gene expression dynamics and location in plants is crucial for applications ranging from basic biological research to agricultural biotechnology (e.g., biofuel development). However, current methods cannot provide in vivo dynamic detection of genomic targets in plants. This limitation is due to the complex sample preparation needed by current methods for nucleic acids detection, which disrupt spatial and temporal resolution. We report the development of a unique multimodal method based on plasmonics-active nanoprobes, referred to inverse molecular sensitnels capable of in vivo imaging and biosensing of microRNA biotargets within whole plant using surface-enhanced Raman scattering (SERS) detection. This work lays the foundations for in vivo functional imaging of RNA biotargets in plants with previously unmet spatial and temporal resolution for many applications ranging from agricultural biotechnology to biofuel research.
Single cell analysis can aid the study of molecular events responsible for cellular functions and unveil their connections to the biological functions of an organism. Biosensors based on surface enhanced Raman spectroscopy (SERS) can be used to this end and offer several advantages over other sensing platforms, such as sensitivity and multiplexed capabilities, among others. While SERS nanosensors/nanoparticles have been used for analysis in single cells, the delivery of such biosensors relies on cellular uptake, which requires long incubation time and has different efficiencies among cell lines. Nanosensors based on tapered optical fibers, instead, can be inserted in single cells and detect target molecules in subcellular compartment. The combination of these sensing devices with the transduction mechanism of nucleic acid based nanoprobes (i.e. inverse molecular sentinels) will permit the more direct detection of nucleic acids within single cells. This paper presents the development of tapered fiber-based biosensors for the detection of nucleic acid biomarkers in plant cells. The use of inverse molecular sentinels in plant cell was demonstrated. Sensors based on tapered fibers were fabricated and used to measure SERS from a single cell.
The development of rapid, easy-to-use and highly sensitive DNA detection methods has received increasing interest for medical diagnostics and research purposes. Our laboratory has developed several chip-based DNA biosensors including molecular sentinel-on-chip (MSC), multiplex MSC, and inverse molecular sentinel-on-chip (iMS-on-Chip). These sensors use surface-enhanced Raman scattering (SERS) plasmonic chips, functionalized with DNA probes for single-step DNA detection. The sensing mechanisms is based on the hybridization of target sequences and DNA probes, resulting in a displacement of a SERS reporter from the chip surface. This distance increase results in change in SERS signal intensity from the reporter, thus indicating the capture, and therefore the presence, of the target nucleic acid sequence. The nucleic acid probes and the SERS chip, which compose the sensing platform, were designed for single-step DNA detection. The target sequences are detected by delivery of a sample solutions on a functionalized chip and characterization of the SERS signals, after 1 - 2 hr incubation. These techniques avoid labeling of the target sequence or washing to remove unreacted components, making them easy-to-use and cost effective. The use of SERS chip for medical diagnostics was demonstrated by detecting genetic biomarkers for respiratory viral infection and the DNA of dengue virus 4.
The development of sensitive and selective biosensing techniques is of great interest for clinical diagnostics. Here, we describe the development and application of a surface enhanced Raman scattering (SERS) sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, for the detection of nucleic acid biomarkers in biological samples. This iMS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform for a homogenous assay for multiplexed detection of nucleic acid biomarkers, including DNA, RNA and microRNA (miRNA). The "OFF-to-ON" signal switch is based on a non-enzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. Here, we demonstrate the development of iMS nanoprobes for the detection of DNA sequences as well as a modified design of the nanoprobe for the detection of short (22-nt) microRNA sequences. The application of iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of nucleic acid biomarkers, including short miRNAs molecules.
The development of rapid, easy-to-use, cost-effective, high accuracy, and high sensitive DNA detection methods for molecular diagnostics has been receiving increasing interest. Over the last five years, our laboratory has developed several chip-based DNA detection techniques including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). In these techniques, plasmonic surface-enhanced Raman scattering (SERS) Nanowave chips were functionalized with DNA probes for single-step DNA detection. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the Nanowave chip’s gold surface. This distance change resulted in change in SERS 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 Nanowave chips and SERS signals were measured after 1h - 2h incubation. 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 techniques for medical diagnostics was illustrated by the detection of genetic biomarkers for respiratory viral infection and of dengue virus 4 DNA.
This lecture presents an overview of recent advances in the development of optical nanobiosensor and nanoprobe technology at the nexus of engineering, biology, medicine and nanotechnology. The presentation describes two areas of research related to the development of nanoprobes and nanosensors for biomolecule detection and single-cell analysis : (1) plasmonics nanoprobes using surface-enhanced Raman scattering (SERS) detection; and (2) nanobiosensors for in vivo analysis of a single cell for molecular diagnostics and imaging, and ultra-high throughput screening. Novel nanobiosensors and nanoprobes combining bio-recognition and nanotechnology have been developed for in-vitro molecular diagnostics and in-vivo monitoring of biological targets and biochemical processes in a single living cell. These studies demonstrate applications of plasmonics “molecular sentinel” nanoprobes for diagnostics of diseases such as cancer and the use of nano-biosensors for measurements of molecular signaling pathways inside a single cell. Fiberoptics-based nanobiosensors are used to detect apoptotic processes in single cells following photodynamic cancer treatment or to monitor pH in cancer cells. These nanodevices open new possibilities to a wide range of applications in medical diagnostics at the point of care, global health, molecular imaging, biology research, ultra-high throughput screening, and investigations of the therapeutic action of pharmaceutical agents.
We have developed a novel label-free approach referred to as plasmonic coupling interference (PCI) nanoprobes for
nucleic acid detection using surface-enhanced Raman scattering (SERS). To induce a strong plasmonic coupling effect
and SERS signal, a nano-network of silver nanoparticles having the Raman label located between adjacent nanoparticles
is assembled by Raman-labeled nucleic acid duplexes. The PCI method then utilizes specific nucleic acid sequences of
interest as competitor elements of the Raman-labeled DNA probes to interfere the formation of the nucleic acid-crosslinked
nano-networks in a competitive binding process, resulting in a reduced SERS signal. We illustrate the potential of
the PCI technique for biomedical applications 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.
This paper describes the use of plasmonics-based nanoprobes for detection of multidrug-resistant tuberculosis gene. The plasmonics nanoprobe is composed of a silver nanoparticle pre-coated with a stem-loop DNA probe that is tagged with a Raman label at one end of the stem region, while the other end of the probe is covalently conjugated to the nanoparticle via a thiol-silver bond. The loop region is designed to detect a specific target gene sequence. In the absence of target, the Raman label is in close proximity to the metal surface, resulting in an intense SERS signal upon laser excitation. In the presence of the target DNA sequence, hybridization between the target and probe disrupts the stem-loop configuration, separating the Raman label from the metal surface and quenching the SERS signal. In this study, we successfully demonstrated for the first time the feasibility of using plasmonics nanoprobes for the detection of multidrug-resistant tuberculosis gene.
A critical aspect of the use of nanoprobes for intracellular studies in chemical and biological sensing involves a fundamental understanding of their uptake and trajectory in cells. In this study, we describe experiments using surface-enhanced Raman scattering (SERS) spectroscopy and mapping to track cellular uptake of plasmonics-active labeled nanoparticles. Three different Raman-active labels with positive, negative, and neutral charges were conjugated to silver colloidal nanoparticles with the aim of spatially and temporally profiling intracellular delivery and tracking of nanoprobes during uptake in single mammalian cells. 1-D Raman spectra and 2-D Raman mapping are used to identify and locate the probes via their SERS signal intensities. Because Raman spectroscopy is very specific for identification of chemical and molecular signatures, the development of functionalized plasmonics-active nanoprobes capable of exploring intracellular spaces and processes has the ability to provide specific information on the effects of biological and chemical pollutants in the intracellular environment. The results indicate that this technique will allow study of when, where, and how these substances affect cells and living organisms.
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