Silent Region Research Articles

Development of a Tri-Functional Nanoprobe for Background-Free SERS Detection of Sialic Acid on the Cell Surface

Sialic acid (SA) on the surface of cells is indispensable in numerous physiological and pathological processes, and sensitive and reproducible detection of SA is crucial for diagnosis and therapy in many diseases. Here, we developed a tri-functional nanoprobe as a sensitive and straightforward surface-enhanced Raman spectroscopy (SERS) nanoprobe for sialoglycan detection on cell surfaces. The reporter was designed to provide three key functionalities that make it ideal for SA detection. First, we employed two recognition groups, phenylboronic acid and an ammonium group, that enhance SA recognition and capture efficiency. Second, we used cyano as the Raman reporter because it emits in the cellular Raman silent region. Finally, thiol acted as an anchoring agent to conjugate the reporter to silver nanocubes to provide SERS enhancement. Our molecular nanoprobe design demonstrated the ability to detect SA on the cell surface with high sensitivity and spatial resolution, opening up new routes to cellular diagnostics.

Vibrational spectroscopy and DFT analysis of 4-cyanophenylhydrazine: A potential SERS probe

4-Cyanophenylhydrazine (4-CPH) is an organic synthesis intermediate. To date, several products derived from 4-CPH have been well studied; however, 4-CPH itself has not been extensively investigated. Herein, we performed vibrational and theoretical analyses of 4-CPH. Density functional theory (DFT) calculations were applied to predict the IR and Raman spectra of 4-CPH, which were compared with the experimental spectra. The calculated and experimental spectral results were in good agreement, except for an abnormal transformation of the protonated 4-CPH cyano group (C≡N), which was observed in the theoretical IR spectrum. Several wavefunction analyses revealed that this transformation was due to the protonation-induced depolarization of the molecule. Moreover, we verified the applicability of 4-CPH as a probe for surface-enhanced Raman spectroscopy (SERS). We observed a pH-dependent shift in the cyano bond frequency within the silent region and determined, as a novel discovery, that this shift was induced by 4-CPH protonation. Our results provide considerable, fundamental information that confirms the potential of 4-CPH as a SERS probe.

Sensitive detection of alkyne-terminated hydrophobic drug by surface-enhanced stimulated Raman scattering in cetyltrimethylammonium bromide-coated gold nanorod suspensions

We demonstrate the sensitive detection of alkyne-terminated ethinylestradiol (EE2) in a cetyltrimethylammonium bromide (CTAB)-coated gold nanorod (AuNR) suspension by surface-enhanced stimulated Raman scattering (SRS). Since alkynes show a distinct Raman peak at the silent wavenumber region, EE2 can be detected by obtaining the alkyne-specific SRS signals. Here we find the CTAB coating uniformly adsorbs the hydrophobic EE2 and enables it to exist in the plasmonic hotspot of AuNRs without the alkyne binding to AuNR surfaces. As a result, ensemble-averaged plasmonic enhancement using a CTAB-coated AuNR suspension improved the detection limit for SRS signals from EE2 by one order of magnitude.

Use of Raman Spectroscopy for Analysis and Detection of Some Sudanese Edible Oils

Vegetable edible oils provide high nutritional and health value in the Sudanese diet. It is also used in the pharmaceutical industry and an essential ingredient in cosmetics. This paper reviews the use of Raman spectroscopy for the analysis, quality and characterization of edible oils, including six types of oils (corn, extra virgin olive, sunflower, factory sesame, presses sesame, and peanut) purchased from local Sudanese stores. The results showed that the spectra of edible oils are similar, but they show some differences that, despite their smallness, allow them to be distinguished from each other. Divide the spectrum into the fingerprint region. The range ranges between (600 and 1800) cm^-1 the silent region", this spectral region is (1800 to 2500) cm^-1, and range is from (2500 to 3400) cm^-1, known as the "high wave number region". Raman spectra also pliable the determination of the degree of saturation and unsaturation of oils. This characteristic is related to the value of iodine, and the degree of unsaturation can be used to classify and approve oils, which is especially useful with high-quality oils in the appearance of the vibration modes at 1155 cm^-1 and 1525 cm^-1. Adulteration of edible oils with cheaper oils is a major concern in the oil industry. The capabilities of a Raman spectrometer were checked to assess the purity of the samples (peanut spectrum and presses sesame spectrum). Raman spectroscopy allowed the examination of secondary components such as sterols, hydrocarbons, terpene alcohols and polyphenols. Raman spectroscopy is used because this innovative method provides fast, non-destructive and reagent-free measurements, samples do not need to be processed and do not require large volumes.

Determination of NADH by Surface Enhanced Raman Scattering Using Au@MB@Ag NPs

Nicotinamide adenine dinucleotide (NADH) is an important coenzyme involved in various metabolic processes of living cells. As an important biomarker, NADH is associated with breast cancer and Alzheimer’s disease. In this paper, silver plated gold core–shell nanoparticles containing Raman signal molecules were synthesised on the basis of bare gold. Using the Raman peak corresponding to the 4-mercaptobenzonitrile (MB) silent region C≡N vibration for quantification, while avoiding competition with the precious metal surface binding site to be measured, it can also be free from the interference of endogenous biomolecules. On the one hand, it can correct the working curve, on the other hand, it can avoid competing with the binding site. Compared with the core–shell structure prepared here, the limit of detection (LOD) for NADH was only 10−5 M for bare gold and the LOD for the core–shell structure prepared on the basis of bare gold was 3.3 × 10−7 M. In terms of correction, with Rhodamine 6G (R6G) as a Raman signalling molecule, the R2 value before SERS detection and correction is only 0.9405, and the R2 value after correction increases to 0.9853. The unique fingerprint peak of SERS was used to realise the quantitative detection of NADH, which realizes the detection of NADH in complex biological samples of serum and provides the possibility for expanding the early diagnosis of breast cancer.

A decade of alkyne-tag Raman imaging (ATRI): applications in biological systems.

Alkyne functional groups have Raman signatures in a region (1800 cm−1 to 2800 cm−1) that is free from interference from cell components, known as the “silent region”, and alkyne signals in this region were first utilized a decade ago to visualize the nuclear localization of a thymidine analogue EdU. Since then, the strategy of Raman imaging of biological samples by using alkyne functional groups, called alkyne-tag Raman imaging (ATRI), has become widely used. This article reviews the applications of ATRI in biological samples ranging from organelles to whole animal models, and briefly discusses the prospects for this technique.

Nucleic Acid Hybridization-Based Noise Suppression for Ultraselective Multiplexed Amplification of Mutant Variants.

The analysis of mutant nucleic acid (NA) variants can provide crucial clinical and biological insights for many diseases. Yet, existing analysis techniques are generally constrained by nonspecific "noise" signals from excessive wildtype background sequences, especially under rapid isothermal multiplexed target amplification conditions. Herein, the molecular hybridization chemistry between NA bases is manipulated to suppress noise signals and achieve ultraselective multiplexed detection of cancer gene fusion NA variants. Firstly, modified locked NA (LNA) bases are rationally introduced into oligonucleotide sequences as designed "locker probes" for high affinity hybridization to wildtype sequences, leading to enrichment of mutant variants for multiplexed isothermal amplification. Secondly, locker probes are coupled with a customized "proximity-programmed" (SERS) readout which allows precise control of hybridization-based plasmonic signaling to specifically detect multiple target amplicons within a single reaction. Moreover, the use of triple bond Raman reporters endows NA noise signal-free quantification in the Raman silent region (≈1800-2600 cm-1 ). With this dual molecular hybridization-based strategy, ultraselective multiplexed detection of gene fusion NA variants in cancer cellular models is actualized with successful noise suppression of native wildtype sequences. The distinct benefits of isothermal NA amplification and SERS multiplexing ability are simultaneously harnessed.

Sandwich-type detection of nucleic acids by bioorthogonal SERS probes

Nucleic acids in body fluids, such as circulating cell-free nucleic acids, viral DNA, and RNA have received much attention for their great potential as biomarkers in liquid biopsies of serious diseases. Although quantitative polymerase chain reaction (qPCR) has been traditionally used as a laboratory-based assay for measuring nucleic acids, there is a strong demand for techniques to qualitatively, rapidly, and simply measure the extremely low-abundance nucleic acids in order to realize the nucleic acid-based liquid biopsies. With this aim in mind, we developed a simple and highly sensitive sandwich-type assay for nucleic acids using a combination of surface-enhanced Raman scattering (SERS), which enhances Raman scattering by 108– to 1010–fold, and bioorthogonal Raman tags, which generate signals in the biologically silent region (1800–2800 cm−1). Using gold nanorods having approximately 240 strands of oligonucleotides and 4-cyano-N-(2-mercaptoethyl)benzamide (4CMB) as the bioorthogonal Raman tag, we successfully detected target nucleic acids in a sequence-selective manner.

Gene repression in S. cerevisiae-looking beyond Sir-dependent gene silencing.

Gene silencing by the SIR (Silent Information Region) family of proteins in S. cerevisiae has been extensively studied and has served as a founding paradigm for our general understanding of gene repression and its links to histone deacetylation and chromatin structure. In recent years, our understanding of other mechanisms of gene repression in S.cerevisiae was significantly advanced. In this review, we focuson such Sir-independent mechanisms of gene repression executed by various Histone Deacetylases (HDACs) and Histone Methyl Transferases (HMTs). We focus on the genes regulated by these enzymes and their known mechanisms of action. We describe the cooperation and redundancy between HDACs and HMTs, and their involvement in gene repression by non-coding RNAs or by their non-histone substrates. We also propose models of epigenetic transmission of the chromatin structures produced by these enzymes and discuss these in the context of gene repression phenomena in other organisms. These include the recycling of the epigenetic marks imposed by HMTs or the recycling of the complexes harboring HDACs.

A confocal Raman microscopic visualization of small penetrants in cellulose acetate using a deuterium-labeling technique

The purpose of the present study was to visualize the sorption dynamics of small compounds, such as propylene glycol (PG) in cellulose acetate (CA) film, by deuterium (d) labeling-aided confocal Raman spectroscopy (CRM). Substitution of hydrogen atoms in the target molecule with deuterium caused a marked shift of C-H bond-related Raman bands to low wavenumbers, while the number of deuterium did not affect the magnitude of the shift. Raman bands derived from the stretching vibration of C–H near 2900 cm−1 for PG and ethanol were shifted to approximately 2100 cm−1 for PG-d6 and ethanol-d5 in the silent region of the CA Raman spectrum. When PG-d6 was dissolved in glycerol triacetate (GTA), the observed Raman intensity ratio at 2123 cm−1 of PG-d6 against 1739 cm−1 of GTA (C=O bond-related) showed a linear relationship between the molar and intensity ratios, indicating that the observed Raman intensity can be used for quantitative assay of the target in polymer film. The depth profiling experiments by CRM revealed that the distribution (or sorption) of PG-d6 in CA film was successfully visualized as a function of Raman band at the characteristic 2123 cm−1 intensity ratio.

Abstract 5267: Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells

Abstract Ponatinib is a clinically approved tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML). Drug resistance is a widespread problem in CML treatment, where ponatinib resistant patients have very limited treatment options. In this study stimulated Raman scattering (SRS) microscopy was used to allow label-free imaging of intracellular ponatinib with high sensitivity and specificity in live human CML cell lines, in the context of ponatinib resistance. Ponatinib has an alkyne moiety in its structure that makes it inherently Raman active in the cellular silent region of the Raman spectrum. SRS microscopy represents a powerful imaging tool for visualizing label-free drug molecules within cells with high resolution, without the need for additional labels, or nanoparticle sensors as used in many other optical imaging technologies. It provides Raman imaging with minimal spectral distortion and a quantitative output, allowing the intracellular concentrations of drug molecules to be accurately determined. Intracellular visualization of ponatinib was achieved at biologically relevant, nanomolar concentrations for the first time using SRS. It was determined that ponatinib is sequestered into the lysosomes, with a higher lysosomal concentration found in drug resistant cells. This was associated with increased lysosome biogenesis. Target engagement studies showed that treatment with chloroquine reduced ponatinib accumulation in lysosomes, but did not re-sensitise cells to ponatinib, confirming BCR-ABL independent resistance mechanism in this CML cell model. In summary, we have visualized intracellular ponatinib localization for the first time using SRS, demonstrating that acquired drug resistance can influence drug uptake and localisation in CML, which in turn has an effect on target engagement. Citation Format: Kristel Sepp, Martin Lee, Marie T. Bluntzer, G. Vignir Helgason, Alison N. Hulme, Valerie G. Brunton. Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5267.

A New Benchmark and an Attribute-Guided Multilevel Feature Representation Network for Fine-Grained Ship Classification in Optical Remote Sensing Images

Maritime activities are essential aspects of human society. Accurate classification of ships is vital for maritime surveillance and meaningful to numerous civil and military applications. However, most studies conducted are limited to the coarse-grained ship classification. Few studies on fine-grained ship classification have been undertaken despite its accuracy and practicability. In this study, we construct a new benchmark for fine-grained ship classification which consists of 23 fine-grained categories of ships. Besides the category label, the benchmark contains several other attribute information. To solve the problem of interclass similarity, an attribute-guided multilevel enhanced feature representation network (AMEFRN) is proposed. Concretely, a multilevel enhanced visual feature representation is designed to fuse the reweighted regional features in order to focus more on the silent region and suppress the other regions. Further to this, considering the complementary role of attribute information in ship identification, an attribute-guided feature extraction branch is proposed, which extracts the auxiliary attribute features by utilizing the attribute information as supervision. Finally, the attribute features and the enhanced visual features jointly function as a feature representation for classification. Compared to other existing classification models, AMEFRN has better performance with an overall accuracy rate of 93.58% on the established fine-grained ship classification dataset. Moreover, it can be easily embedded into most CNN models as well as trained end-to-end.

Target-triggered hot spot dispersion for cellular biothiol detection via background-free surface-enhanced Raman scattering tags

Abnormal cellular biothiol levels are related to many abnormal physiological processes, including cancer, multidrug resistance and Alzheimer's disease, etc. In this study, the nano-aggregates of the background-free surface-enhanced Raman scattering (SERS) tags were constructed and developed for the intracellular biothiol detection via a target-triggered disaggregation process. The plasmonic nano-tags were prepared by coating gold nanoparticles with a Raman reporter (4-mercaptobenzonitrile, MBN), which exhibits a single strong peak in the cellular Raman silent region (1800−2800 cm−1) that can eliminate the background interference of cells. Interestingly, this reporter is also the host ligand for guest mercury ions. The coordination of mercury/cyano group induce the formation of the pre-aggregates of nano-tags and the formed nano-aggregates allowing strong SERS signals of reporters. Intracellular biothiols show higher affinity to mercury ions than the SERS tags do, which can break the hot spot geometry and redisperse tags by taking away mercury ions from nano-aggregates, which dramatically decreases the SERS signals of reporters previously laid on gold nanoparticles. The developed SERS “turn off” method was used for biothiol detections in normal, cancer, drug-resistant cells, and biothiol dynamics during chemotherapy. The results demonstrate that the drug-resistant cells (MCF-ADR) lie in a higher biothiol level than cancer cells (MCF-7 and HepG2), and the normal cells (LO2) give a lower biothiol concentration compared with cancer cells. Moreover, most cancer cells are more sensitive to doxorubicin compared with the normal ones. This study provides an important strategy in learning the cellular processes that are highly associated with intracellular biothiol level.

When Prussian Blue Meets Porous Gold Nanoparticles: A High Signal-to-Background Surface-Enhanced Raman Scattering Probe for Cellular Biomarker Imaging.

Profiling cellular biomarkers without the interference of endogenous signals could facilitate the investigation of complex intracellular biological events and provide new possibilities for precision disease diagnosis. Herein, a surface-enhanced Raman scattering (SERS) probe with a high signal-to-background ratio (SBR) for cellular biomarker imaging is constructed. The probes are prepared by incorporating Prussian blue (PB) with porous gold nanoparticles (p-Au NPs). Due to their rich built-in Raman hotspots, the p-Au NPs are excellent SERS substrates that can significantly amplify the signals of the incorporated PB. In parallel, PB shows a single peak in the cellular silent region, where the signals from the probes and endogenous molecules can be completely resolved without the need of complex spectral unmixing. As a consequence, the combination of probe signal enhancement and background elimination endows the SERS probes with an extremely high SBR. To evaluate their performance in biomarker imaging, the high-SBR SERS probes are utilized to profile folic acids at a single-cell level. This background-free, high-precision imaging technique is conducive to early diagnosis and therapeutic response of cancer that is of great importance in clinical settings.

451 Identification and characterization of euchromatic regions associated with gene expression and intramuscular fat in Nelore cattle.

In eukaryotes, DNA is organized along with histones in nucleoprotein complexes known as chromatin, which has nucleosomes as their fundamental unit. Chromatin exists in two forms: euchromatin, corresponding to a lightly condensed structure and an easily transcribed region, and heterochromatin, a highly condensed and transcriptionally silent region. Therefore, the chromatin compaction degree interferes with regulation of gene expression, and nucleosomes act as gene silencers. In this context, using Assay for Transposase-Accessible Chromatin technique (ATAC-seq), the aims of the present study were to identify, map and characterize euchromatin regions in Longissimus dorsi muscle of Nellore cattle to understand if these regions are associated with gene expression and intramuscular fat content. Different transposase-treated nuclei concentrations were tested: 50 thousand, 75 thousand and 100 thousand, and their nucleosomal periodicity was analyzed to check the efficiency of ATAC-seq enzymatic activity. From these, 6,811, 11,121, and 11,473 euchromatin peaks were respectively found by using Model-based Analysis of ChIP-Seq (MACS2). A total of 6,212 open chromatin regions were coincident among the three nuclei concentrations, and this data was used in subsequent analyzes. Enrichment analysis performed using package RegioneR in R, considering significance of p < 0.05, revealed over-representation of peaks in 1) transcription start sites of genes expressed in skeletal muscle; 2) differentially expressed genes (GDE) associated with intramuscular fat content; 3) expression quantitative trait loci (eQTL) identified in skeletal muscle tissue. Our results indicate that skeletal muscle regulatory regions identified by ATAC-seq are involved in the control of gene expression and intramuscular fat content in cattle.

Broadband ultrasound-trapping barrier based on hollow cylinder with a periodic grating

It is important to modulate ultrasonic waves in some ultrasonic application fields. We formed an ultrasound-trapping barrier through a hollow cylinder with a periodic grating on the surface and simulated the barrier using the finite element method. Using the Schlieren imaging technique, we visualized the ultrasound field distribution in the structure. Both the experimental and simulation results revealed that the structure can work in a broadband frequency range, demonstrating the effectiveness and practicability of the structure as a virtual ultrasound barrier. We also describe how the silent region and the ultrasound intensity can be adjusted in the hollow cylinder.

SERS Nanoprobes in Biologically Raman Silent Region for Tumor Cell Imaging and In Vivo Tumor Spectral Detection in Mice

AbstractSurface enhanced Raman scattering (SERS) is widely used in biological applications due to its high sensitivity and deep tissue penetration. Most SERS nanoprobes with fluorophore attachments have Raman signals between 1400 and 1700 cm−1. Here, a series of SERS nanoprobes anchored with alkyne moieties in the biologically Raman‐silent region through CAu bond formation is prepared. To achieve target‐specific recognition, two nucleic acid aptamers (AS1411 and MUC1) and two control oligonucleotides (C‐con and T‐con) with the same alkyne moiety are also attached on the Au surface, respectively. Both aptamer‐charged SERS nanoprobes successfully target MCF‐7 cells, which could possibly achieve cross‐check of target cells and overcome the possible false positive results. And the limit of detection reaches up to five cancer cells, which has great potential in circulating tumor cells detection. Further in vivo studies indicate that both SERS nanoprobes are successfully employed to target tumors in living mice with an obvious signal (≈2205 cm−1) at the Raman silent region after tail intravenous injection for the first time.

A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat.

Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.

Metal Carbonyls for the Biointerference-Free Ratiometric Surface-Enhanced Raman Spectroscopy-Based Assay for Cell-Free Circulating DNA of Epstein-Barr Virus in Blood.

By taking advantage of the spectral properties of metal carbonyls, we have designed a surface-enhanced Raman spectroscopy (SERS) ratiometric assay for measuring cell-free circulating DNA (cfDNA) from Epstein-Barr virus in blood for nasopharyngeal carcinoma (NPC). This assay consists of a rhenium carbonyl (Re-CO) to serve as a DNA probe, an osmium carbonyl (Os-CO) embedded within the SERS-active substrate as an internal reference, and a streptavidin layer on the surface of the substrate. Hybridization of cfDNA with biotinylated-capture sequence leads to immobilization of cfDNA on the substrate. The binding of Re-CO via daunorubicin (DNR) to cfDNA is accompanied by an appearance of a strong symmetry stretching vibrations peak at 2113 cm-1, which has spectral overlap with Os-CO (2025 cm-1). This results in an increase in the I2113/ I2025 ratio and quantitatively correlates with cfDNA. This SERS assay can be readily used to detect cfDNA in blood samples from patients due to the intensity ratio of I2113/ I2025 lying in a silent region (1780-2200 cm-1) in the SERS spectrum of the biomolecules.

Alkyne-DNA-Functionalized Alloyed Au/Ag Nanospheres for Ratiometric Surface-Enhanced Raman Scattering Imaging Assay of Endonuclease Activity in Live Cells.

A novel ratiometric surface-enhanced Raman scattering (SERS) nanosensor has been developed to probe the activity of endonuclease under in vitro and in living cells conditions. The optimized alloyed Au/Ag nanoparticles (NPs) were synthesized as the SERS substrate, which combined the superior properties of both pure Au and pure Ag nanoparticles: they exhibit excellent plasmonic property with high chemical stability and low cytotoxicity. They were then employed for quantitative detection of endonuclease through functionalization with single-stranded DNA (ssDNA) carrying 3-[4-(phenylethynyl)benzylthio]propanoic acid (PEB) as endonuclease-responsive SERS signaling molecule and 4-thiophenylacetylene (TPA) as the internal standard SERS signaling molecule. In the presence of endonuclease, the ssDNA was cleaved, releasing PEB molecules from the particle surface and decreasing the SERS signal at 2215 cm-1 from PEB. Since the SERS signal at 1983 cm-1 from alkynyl TPA remained the same, quantitative detection of endonuclease was achieved, based on the ratiometric peak intensity of I1983/ I2215, with a detection limit as low as 0.056 unit/mL. A highly biocompatible and antijamming ratiometric SERS sensor was established by combining the alloyed Au/AgNPs with two unique alkynes molecules with Raman signals in the cellular silent region. The ratiometric sensor was successfully employed to detect intracellular endonuclease activity as well as endonuclease in living cells for the first time.