Study of nusinersen metabolites in the cerebrospinal fluid of children with spinal muscular atrophy using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Three different sample preparation methods were tested for extraction and purification, but solid phase extraction appeared to be the most suitable, allowing a significant sample enrichment (40-fold). This step was necessary to detect and identify metabolites of nusinersen in the cerebrospinal fluid. The developed and applied analytical procedure enabled the identification of nusinersen metabolites: sequences shorter by several nucleotides from the 3' end; shorter by several nucleotides from both the 3' and 5' ends; and some depurination products. To the best of our knowledge, this is the first report on the analysis and identification of nusinersen metabolites in cerebrospinal fluid samples taken from children with spinal muscular atrophy treated with Spinraza.

Elimination of Toxic Solvents from Analytical Methods in Food Analysis: Caffeine Determination in Tea as an Example.

This study presents an innovative method for caffeine determination in tea, employing ethanol as the sole organic solvent for both SPE sample preparation and chromatographic analysis. This approach aligns with green chemistry principles, as confirmed by a comparative study highlighting ethanol's safety and eco-friendliness compared to traditional solvents. The experiments validate ethanol's efficacy in caffeine extraction and chromatographic analysis, minimizing environmental impact and eliminating toxicity risks. Utilizing a reduced chromatography column enhances the method's efficiency and sustainability, resulting in a low limit of quantitation (0.125 µg/mL) and good reproducibility (RSD < 2.5%). Based on tea from the Polish market, the findings reveal the caffeine content (19.29-37.69 mg/g) and endorse ethanol's role in enhancing sustainable chemical analysis in food science.

Effect of Non-Modified as Well as Surface-Modified SiO2 Nanoparticles on Red Blood Cells, Biological and Model Membranes.

Nanoparticles are extremely promising components that are used in diagnostics and medical therapies. Among them, silica nanoparticles are ultrafine materials that, due to their unique physicochemical properties, have already been used in biomedicine, for instance, in cancer therapy. The aim of this study was to investigate the cytotoxicity of three types of nanoparticles (SiO2, SiO2-SH, and SiO2-COOH) in relation to red blood cells, as well as the impact of silicon dioxide nanoparticles on biological membranes and liposome models of membranes. The results obtained prove that hemolytic toxicity depends on the concentration of nanoparticles and the incubation period. Silica nanoparticles have a marginal impact on the changes in the osmotic resistance of erythrocytes, except for SiO2-COOH, which, similarly to SiO2 and SiO2-SH, changes the shape of erythrocytes from discocytes mainly towards echinocytes. What is more, nanosilica has an impact on the change in fluidity of biological and model membranes. The research gives a new view of the practical possibilities for the use of large-grain nanoparticles in biomedicine.

Demographic and clinical characteristics of patients who recommence clozapine following therapy interruptions.

OBJECTIVE: The proportion of patients who recommence clozapine after cessation, the time taken to resume clozapine post-cessation, and distinguishing demographic and clinical characteristics of this group have been poorly researched. We evaluated these in the current study. METHOD: We retrospectively extracted selected demographic and clinical variables and clozapine treatment interruption and recommencement data up to December 2018 of a cohort of 458 patients who first commenced clozapine between 2006 and 2016. The study was conducted at three Australian health services. RESULTS: Of the 310 (69%) patients who had at least one interruption of clozapine treatment, 170 (54.8%) did not resume clozapine, and 140 (45.2%) recommenced it after the first interruption. More than half of those who recommenced did so within a month and 80% by 12 months. Cox regression analysis revealed that age was significantly associated with recommencement, with a 2% decrease in the likelihood of restarting after an interruption for each year later that clozapine was initially commenced (HR = 0.98 95%CI: 0.97, 0.997, p = 0.02). Those who ceased clozapine due to adverse effects were less likely to restart than those who ceased due to noncompliance (HR = 0.63 95%CI: 0.41, 0.97, p = 0.03). More time on clozapine prior to interruption increased the likelihood of restarting it, with each additional month on clozapine increasing this likelihood by 1% (HR = 1.01 95%CI: 1.01, 1.02, p < 0.001). CONCLUSION: If the distinguishing demographic and clinical characteristics of the group identified in this study are corroborated through further research, this could further validate the need to identify treatment resistance and commence clozapine early in people with schizophrenia and provide appropriate interventions to those more at risk of permanent discontinuation of clozapine.

Intracellular pH - Advantages and pitfalls of surface-enhanced Raman scattering and fluorescence microscopy - A review.

The value of pH in various parts of protoplasm can affect nearly all aspects of cell functions. Therefore, the determination of intracellular acid-base features is required in many areas of biological and biochemical studies. Because of a significant scientific importance of in vivo intracellular pH measurements, various groups carried out such experiments. In this review article we describe intracellular pH measurements using two the most sensitive optical spectroscopies: surface-enhanced Raman scattering (SERS) and fluorescence. It is reasonable to present these two techniques in one review article because the experimental approach in Raman and fluorescence experiments is relatively similar. The basic theoretical background explaining the mechanism of operation of fluorescence and SERS sensors are discussed and the motivations to carry out intracellular pH measurements are briefly described. Future perspectives in this field are also discussed.

Surface Enhanced Raman Spectroscopy for DNA Biosensors-How Far Are We?

A sensitive and accurate identification of specific DNA fragments (usually containing a mutation) can influence clinical decisions. Standard methods routinely used for this type of detection are PCR (Polymerase Chain Reaction, and its modifications), and, less commonly, NGS (Next Generation Sequencing). However, these methods are quite complicated, requiring time-consuming, multi-stage sample preparation, and specially trained staff. Usually, it takes weeks for patients to obtain their results. Therefore, different DNA sensors are being intensively developed by many groups. One technique often used to obtain an analytical signal from DNA sensors is Raman spectroscopy. Its modification, surface-enhanced Raman spectroscopy (SERS), is especially useful for practical analytical applications due to its extra low limit of detection. SERS takes advantage of the strong increase in the efficiency of Raman signal generation caused by a local electric field enhancement near plasmonic (typically gold and silver) nanostructures. In this condensed review, we describe the most important types of SERS-based nanosensors for genetic studies and comment on their potential for becoming diagnostic tools.

SERS Studies of Adsorption on Gold Surfaces of Mononucleotides with Attached Hexanethiol Moiety: Comparison with Selected Single-Stranded Thiolated DNA Fragments.

The attachment of DNA strands to gold surfaces is performed in many devices, such as various DNA sensors. One of the standard methods used to immobilize DNA on gold surfaces involves two steps: the attachment of a thiol linker group (usually in the form of alkanethiol moiety) to the DNA strand, and the chemical reaction between the thiol-terminated DNA and the gold surface. Since thiols react chemically with the surface of gold substrates, forming very stable Au-S bonds, it is often assumed that the chemisorption on the gold surface of nucleotides with an attached thiol linker group leads to the formation of an order layer with the linking moieties relatively densely packed on the gold surface. In this contribution we show that chemisorption of thiolated mononucleotides does not occur according to this model. For example, the thiolated mononucleotide containing adenine strongly interacts with the gold surface via the adenine moiety. Moreover, bonding of the mononucleotide containing adenine to the gold surface is relatively similar to the bonding of adenine, and the main difference is that the adenine interacts with the gold surface mainly through the pyrimidine ring, while for adenine mononucleotide interaction via the imidazole ring also significantly contributes to the total bonding. A similar effect was observed for the mononucleotide containing cytosine, and the main difference between the interaction with the gold surface of cytosine and cytosine mononucleotide is that mononucleotide containing cytosine interacts with the gold surface to a significantly larger extend via the carboxylic group of the base. We also show that the structure of the layer formed on the gold surface by the thiolated mononucleotides may be significantly different than the structure of the layer formed by thiolated single-stranded DNA containing even as few as two bases.

Graphene and Graphene Oxide Applications for SERS Sensing and Imaging.

Surface Enhanced Raman Spectroscopy (SERS) has a long history as an ultrasensitive platform for the detection of biological species from small aromatic molecules to complex biological systems as circulating tumor cells. Thanks to unique properties of graphene, the range of SERS applications has largely expanded. Graphene is efficient fluorescence quencher improving quality of Raman spectra. It contributes also to the SERS enhancement factor through the chemical mechanism. In turn, the chemical flexibility of Reduced Graphene Oxide (RGO) enables tunable adsorption of molecules or cells on SERS active surfaces. Graphene oxide composites with SERS active nanoparticles have been also applied for Raman imaging of cells. This review presents a survey of SERS assays employing graphene or RGO emphasizing the improvement of SERS enhancement brought by graphene or RGO. The structure and physical properties of graphene and RGO will be discussed too.

Influence of amine and thiol modifications at the 3' ends of single stranded DNA molecules on their adsorption on gold surface and the efficiency of their hybridization.

Adsorption of molecules of DNA (deoxyribonucleic acid) or modified DNA on gold surfaces is often the first step in construction of many various biosensors, including biosensors for detection of DNA with a particular sequence. In this work we study the influence of amine and thiol modifications at the 3' ends of single stranded DNA (ssDNA) molecules on their adsorption on the surface of gold substrates and on the efficiency of hybridization of immobilized DNA with the complementary single stranded DNA. The characterization of formed layers has been carried out using infrared spectroscopy and atomic force microscopy. As model single stranded DNA we used DNA containing 20 adenine bases, whereas the complementary DNA contained 20 thymine bases. We found that the bands in polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS) spectra of layers formed from thiol-modified DNA are significantly narrower and sharper, indicating their higher regularity in the orientation of DNA on gold surface when using thiol linker. Also, hybridization of the layer of thiol-modified DNA containing 20 adenine bases with the respective DNA containing thymine bases leads to formation of much more organized structures than in the case of unmodified DNA or DNA with the amine linker. We conclude that the thiol-modified ssDNA is more promising for the preparation of biosensors, in comparison with the amine-modified or unmodified ssDNA. We have also found that the above-mentioned modifications at the 3' end of ssDNA significantly influence the IR spectrum (and hence the structure) of polycrystalline films formed from such compounds, even though adsorbed fragments contain less than 5% of the DNA chain. This effect should be taken into account when comparing IR spectra of various polycrystalline films formed from modified and unmodified DNA.

Potential of Surface Enhanced Raman Spectroscopy (SERS) in Therapeutic Drug Monitoring (TDM). A Critical Review.

Surface-Enhanced Raman Spectroscopy (SERS) is a label-free technique that enables quick monitoring of substances at low concentrations in biological matrices. These advantages make it an attractive tool for the development of point-of-care tests suitable for Therapeutic Drug Monitoring (TDM) of drugs with a narrow therapeutic window, such as chemotherapeutic drugs, immunosuppressants, and various anticonvulsants. In this article, the current applications of SERS in the field of TDM for cancer therapy are discussed in detail and illustrated according to the different strategies and substrates. In particular, future perspectives are provided and special concerns regarding the standardization of self-assembly methods and nanofabrication procedures, quality assurance, and technology readiness are critically evaluated.

Evaluation of Anthelmintic Activity and Composition of Pumpkin (Cucurbita pepo L.) Seed Extracts-In Vitro and in Vivo Studies.

UNLABELLED: A significant number of studies report growing resistance in nematodes thriving in both humans and livestock. This study was conducted to evaluate the in vitro and in vivo anthelmintic efficiency of Curcubita pepo (C. pepo) L. hot water extract (HWE), cold water extract (CWE) or ethanol extract (ETE) on two model nematodes: Caenorhabditis elegans (C. elegans) and Heligmosoides bakeri (H. bakeri). METHODS: Raman, IR and LC-MS spectroscopy analyses were performed on the studied plant material to deliver qualitative and quantitative data on the composition of the obtained extracts: ETE, HWE and CWE. The in vitro activity evaluation showed an impact of C. pepo extracts on C. elegans and different developmental stages of H. bakeri. The following in vivo experiments on mice infected with H. bakeri confirmed inhibitory properties of the most active pumpkin extract selected by the in vitro study. All of the extracts were found to contain cucurbitine, aminoacids, fatty acids, and-for the first time-berberine and palmatine were identified. All C. pepo seed extracts exhibited a nematidicidal potential in vitro, affecting the survival of L1 and L2 H. bakeri larvae. The ETE was the strongest and demonstrated a positive effect on H. bakeri eggs hatching and marked inhibitory properties against worm motility, compared to a PBS control. No significant effects of pumpkin seed extracts on C. elegans integrity or motility were found. The EtOH extract in the in vivo studies showed anthelmintic properties against both H. bakeri fecal egg counts and adult worm burdens. The highest egg counts reduction was observed for the 8 g/kg dose (IC50 against H. bakeri = 2.43; 95% Cl = 2.01-2.94). A decrease in faecal egg counts (FEC) was accompanied by a significant reduction in worm burden of the treated mice compared to the control group. CONCLUSIONS: Pumpkin seed extracts may be used to control of Gastrointestinal (G.I.) nematode infections. This relatively inexpensive alternative to the currently available chemotherapeutic should be considered as a novel drug candidate in the nearest future.

Raman microscopy as a novel tool to detect endothelial dysfunction.

Raman microscopy, a label-free method with high spatial resolution, shows growing potential in various fields of medical diagnostics. Several proof-of-concept studies related to the application of Raman microscopy to detect endothelial dysfunction are summarized in this work. Both ex vivo measurements of the tissues in the murine models of endothelial pathologies, as well as in vitro investigations of the cell cultures in the context of cellular transport, drug action and inflammation processes are discussed. The future directions in application of Raman spectroscopy-based methods in such studies are also described.

Rhodamine 6G conjugated to gold nanoparticles as labels for both SERS and fluorescence
studies on live endothelial cells.

Fluorescence and surface-enhanced Raman scattering (SERS) spectroscopy were employed to investigate the cellular uptake of rhodamine 6G (R6G) alone and of R6G loaded with gold nanoparticles (AuNPs) by endothelial cells. R6G plays the role of a Raman reporter in SERS but also displays strong fluorescence. The presence of bare R6G molecules and R6G-AuNPs in the cytoplasm of the cells is detected via the 2D fluorescence of the dye after a 0.5 h of the incubation with R6G and R6G-AuNPs, and then the concentration of the dye increases within 4 h of exposure. The examination of the cellular uptake of the R6G and R6G-AuNPs species at different temperatures suggests that the internalization of the R6G-AuNPs into endothelial cells occurs mainly via endocytosis. 3D fluorescence imaging of R6G inside cells reveals inhomogeneous distribution of the dye in the cytoplasm. The SERS signal of the Raman reporter inside the cell disappears after 2 h of incubation with R6G-AuNPs and then amino acid residues, purines and pyrimidines become SERS-active via their interactions with the gold. The results highlight the significance of using multiple techniques to cover a spectrum of issues in the application of SERS nanosensors for probing an intracellular environment under comparable and standardized conditions. FigureCellular uptake of bare rhodamine 6G and rhodamine 6G adsorbed onto AuNPs were studied on endothelial cells using fluorescence and surface-enhanced Raman spectroscopy. The internalization of R6G-AuNPs occurs via endocytosis and diffusion resulting in uneven distribution in the cytoplasm.

SERS-based monitoring of the intracellular pH in endothelial cells: the influence of the extracellular environment and tumour necrosis factor-α.

The intracellular pH plays an important role in various cellular processes. In this work, we describe a method for monitoring of the intracellular pH in endothelial cells by using surface enhanced Raman spectroscopy (SERS) and 4-mercaptobenzoic acid (MBA) anchored to gold nanoparticles as pH-sensitive probes. Using the Raman microimaging technique, we analysed changes in intracellular pH induced by buffers with acid or alkaline pH, as well as in endothelial inflammation induced by tumour necrosis factor-α (TNFα). The targeted nanosensor enabled spatial pH measurements revealing distinct changes of the intracellular pH in endosomal compartments of the endothelium. Altogether, SERS-based analysis of intracellular pH proves to be a promising technique for a better understanding of intracellular pH regulation in various subcellular compartments.

A comparison between adsorption mechanism of tricyclic antidepressants on silver nanoparticles and binding modes on receptors. Surface-enhanced Raman spectroscopy studies.

A series of the tricyclic antidepressants known as a surface-active drugs, has been used as a model for an evaluation of their adsorption mechanism on the metal substrate and its relationship to pharmacological action of the chosen drugs. In these studies, six antidepressants were adsorbed on the metal substrate in a form of silver nanoparticles (ca. 30 nm in diameter) and afterwards their interactions have been examined in terms of surface-enhanced Raman spectroscopy (SERS). An analysis of SERS spectra has revealed that the dibenzopine moiety is a primary site of the adsorption with some differences in the orientation with respect to the metal among the studied molecules. The spectral changes due to the interactions with the silver particles also appear in the region typical for vibrations of the side chain. These observations are consistent with a model, in which the tricyclic ring is docked in the outer vestibule of biogenic amine transporters whereas the dimethyl-aminopropyl side chain is pointed to the substrate binding site. This work sheds a light on a potential of SERS technique in predicting a key functional group responsible for drug action.

An impact of the ring substitution in nicorandil on its adsorption on silver nanoparticles. Surface-enhanced Raman spectroscopy studies.

The substituent effect on structure and surface activity of biologically active nicorandil was investigated by means of surface-enhanced Raman spectroscopy (SERS). Vibrational characterization was a basis for investigation of the adsorption profile of nicorandil and 1-methylnicorandil on silver nanoparticles. An assignment of SERS bands was performed by the comparison of the Raman spectra of both compounds in the solid state and in solutions, complemented by DFT calculations. Even though the nitro group was found to be the most attractive to the silver surface, the strong impact of the methyl substituent changed this preferable adsorption mechanism in 1-methylnicorandil. Protonation of the nitrogen atom in the pyridinium ring was also found to have an impact on absorption mechanism.

(-)-R-mevalonolactone studied by ROA and SERS spectroscopy.

Here we present for the first time the experimental and theoretical (DFT/B3LYP/6-311++G**) Raman optical activity (ROA) spectra of (-)-R-mevalonic acid as the δ-lactone form in neat liquid and in the aqueous solution. Quantum chemical calculations show the conformational diversity of (-)-R-mevalonolactone originated from small energy differences between the various conformation of the six-membered ring and the arrangement of the hydroxyl group. According to calculations, the investigated compound takes predominantly the chair conformation with the hydroxyl group in axial position, but the contribution of the other chair and boat conformers in the equilibrium at room temperature is not negligible. Additionally, we present normal Raman and the surface enhanced Raman (SERS) spectra of (-)-R-mevalonolactone adsorbed on the colloidal silver.

Evaluation of the potential of surface enhancement Raman spectroscopy for detection of tricyclic psychotropic drugs. Case studies on imipramine and its metabolite.

The potential use of surface Raman enhanced spectroscopy (SERS) for confirmatory identification and the semi-quantitative analysis of selected tricyclic antidepressants (TCAs) is examined utilizing a conventional silver colloid. Raman and SERS spectra of aqueous solutions of imipramine (Imi) and its metabolite, desipramine (Des), were recorded as the function of concentration using NIR excitation. A good linear correlation is observed for the dependence of the SERS signal at 684 cm(-1) (R(2) = 0.9997) on Imi concentration over the range of 0.75-7.5 µM. The limit of detection of imipramine in the silver colloidal solution is 0.98 µM. SERS spectra of Imi and Des were also recorded for blood plasma samples without prior purification as well as after the use of standard solid phase extraction. All spectra show the characteristic spectral profile of the molecules and moreover, stronger signal enhancement is observed for Imi in the "raw" samples as opposed to Imi extracted from a biological matrix.