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- 4538 to 4547
- Medium: X
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- National Science Foundation
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null (Ed.)Glass nanopipettes have shown promise for applications in single-cell manipulation, analysis, and imaging. In recent years, plasmonic nanopipettes have been developed to enable surface-enhanced Raman spectroscopy (SERS) measurements for single-cell analysis. In this work, we developed a SERS-active nanopipette that can be used to perform long-term and reliable intracellular analysis of single living cells with minimal damage, which is achieved by optimizing the nanopipette geometry and the surface density of the gold nanoparticle (AuNP) layer at the nanopipette tip. To demonstrate its ability in single-cell analysis, we used the nanopipette for intracellular pH sensing. Intracellular pH (pH i ) is vital to cells as it influences cell function and behavior and pathological conditions. The pH sensitivity was realized by simply modifying the AuNP layer with the pH reporter molecule 4-mercaptobenzoic acid. With a response time of less than 5 seconds, the pH sensing range is from 6.0 to 8.0 and the maximum sensitivity is 0.2 pH units. We monitored the pH i change of individual HeLa and fibroblast cells, triggered by the extracellular pH (pH e ) change. The HeLa cancer cells can better resist pH e change and adapt to the weak acidic environment. Plasmonic nanopipettes can be further developed to monitor other intracellular biomarkers.more » « less
Surface-enhanced Raman spectroscopy (SERS) is an attractive method for bio-chemical sensing due to its potential for single molecule sensitivity and the prospect of DNA composition analysis. In this manuscript we leverage metal specific chemical enhancement effect to detect differences in SERS spectra of 200-base length single-stranded DNA (ssDNA) molecules adsorbed on gold or silver nanorod substrates, and then develop and train a linear regression as well as neural network models to predict the composition of ssDNA. Our results indicate that employing substrates of different metals that host a given adsorbed molecule leads to distinct SERS spectra, allowing to probe metal-molecule interactions under distinct chemical enhancement regimes. Leveraging this difference and combining spectra from different metals as an input for PCA (Principal Component Analysis) and NN (Neural Network) models, allows to significantly lower the detection errors compared to manual feature-choosing analysis as well as compared to the case where data from single metal is used. Furthermore, we show that NN model provides superior performance in the presence of complex noise and data dispersion factors that affect SERS signals collected from metal substrates fabricated on different days.
DNA‐templated silver nanoclusters (AgNC@DNA) are a novel type of nanomaterial with advantageous optical properties. Only a few atoms in size, the fluorescence of nanoclusters can be tuned using DNA overhangs. In this study, we explored the properties of AgNCs manufactured on a short single‐stranded (dC)12when adjacent G‐rich sequences (dGN, with
N= 3–15) were added. The ‘red’ emission of AgNC@dC12with λMAX = 660 nm dramatically changed upon the addition of a G‐rich overhang with NG = 15. The pattern of the emission–excitation matrix (EEM) suggested the emergence of two new emissive states at λMAX = 575 nm and λMAX = 710 nm. The appearance of these peaks provides an effective way to design biosensors capable of detecting specific nucleic acid sequences with low fluorescence backgrounds. We used this property to construct an NA‐based switch that brings AgNC and the G overhang near one another, turning ‘ON’ the new fluorescence peaks only when a specific miRNA sequence is present. Next, we tested this detection switch on miR‐371, which is overexpressed in prostate cancer. The results presented provide evidence that this novel fluorescent switch is both sensitive and specific with a limit of detection close to 22 picomoles of the target miR‐371 molecule.
Nucleic acid biosensing technologies have the capability to provide valuable information in applications ranging from medical diagnostics to environmental sensing. The unique properties of plasmonic metallic nanoparticles have been used for sensing purposes and among them, plasmonic sensors based on surface-enhanced Raman scattering (SERS) offer the advantages of sensitive and muliplexed detection owing to the narrow bandwidth of their characteristic Raman spectral features. This paper describes current applications that employ the unique SERS-based inverse molecular sentinel (iMS) nanobiosensors developed in our laboratory. Herein, we demonstrate the use of label-free iMS nanoprobes for detecting specific nucleic acid biomarkers in a wide variety of applications from cancer diagnostics to genetic monitoring for plant biology in renewable biofuel research.
The investigation of complex biological processes requires effective tools for probing the spatiotemporal dynamics of individual cells. Single-cell gene expression analysis, such as RNA in situ hybridization and single-cell PCR, has been demonstrated in various biological applications (Tautz and Pfeifle, Chromosoma 98(2):81–5, 1989; Stahlberg and Bengtsson, Methods 50(4):282–288, 2010; Sanchez-Freire et al., Nat Protoc 7(5):829–838, 2012). However, existing techniques require cell lysis or fixation. The dynamic information and spatiotemporal regulation of the biological process cannot be obtained with these methods. Real-time gene expression analysis in living cells remains an outstanding challenge in the field. Here, we described a single-cell gene expression analysis method in living mammalian cells using a locked nucleic acid/DNA (LNA/DNA) nanobiosensor. This LNA/DNA nanobiosensor consists of a fluorophore-labeled detecting strand and a quenching strand. The fluorophore-labeled LNA probe is designed to hybridize with the target microRNA (miRNA) specifically and displace from the quenching strand, allowing the fluorophore to fluorescence. Large-scale single-cell dynamic gene expression monitoring can be performed using time-lapse microscopy to study spatiotemporal distribution and heterogeneity in gene expression. Multiplex detection of miRNAs can be achieved using different fluorophore-labeled LNA/DNA nanobiosensors. This LNA/DNA protocol is fast, generally applicable, and easily accessible.more » « less