Combined miRNA and SERS urine liquid biopsy for the point-of-care diagnosis and molecular stratification of bladder cancer.

BACKGROUND: Bladder cancer (BC) has the highest per-patient cost of all cancer types. Hence, we aim to develop a non-invasive, point-of-care tool for the diagnostic and molecular stratification of patients with BC based on combined microRNAs (miRNAs) and surface-enhanced Raman spectroscopy (SERS) profiling of urine. METHODS: Next-generation sequencing of the whole miRNome and SERS profiling were performed on urine samples collected from 15 patients with BC and 16 control subjects (CTRLs). A retrospective cohort (BC = 66 and CTRL = 50) and RT-qPCR were used to confirm the selected differently expressed miRNAs. Diagnostic accuracy was assessed using machine learning algorithms (logistic regression, naïve Bayes, and random forest), which were trained to discriminate between BC and CTRL, using as input either miRNAs, SERS, or both. The molecular stratification of BC based on miRNA and SERS profiling was performed to discriminate between high-grade and low-grade tumors and between luminal and basal types. RESULTS: Combining SERS data with three differentially expressed miRNAs (miR-34a-5p, miR-205-3p, miR-210-3p) yielded an Area Under the Curve (AUC) of 0.92 ± 0.06 in discriminating between BC and CTRL, an accuracy which was superior either to miRNAs (AUC = 0.84 ± 0.03) or SERS data (AUC = 0.84 ± 0.05) individually. When evaluating the classification accuracy for luminal and basal BC, the combination of miRNAs and SERS profiling averaged an AUC of 0.95 ± 0.03 across the three machine learning algorithms, again better than miRNA (AUC = 0.89 ± 0.04) or SERS (AUC = 0.92 ± 0.05) individually, although SERS alone performed better in terms of classification accuracy. CONCLUSION: miRNA profiling synergizes with SERS profiling for point-of-care diagnostic and molecular stratification of BC. By combining the two liquid biopsy methods, a clinically relevant tool that can aid BC patients is envisaged.

Fermi Level Equilibration at the Metal-Molecule Interface in Plasmonic Systems.

We highlight a new metal-molecule charge transfer process by tuning the Fermi energy of plasmonic silver nanoparticles (AgNPs) in situ. The strong adsorption of halide ions upshifts the Fermi level of AgNPs by up to ∼0.3 eV in the order Cl- < Br- < I-, favoring the spontaneous charge transfer to aligned molecular acceptor orbitals until charge neutrality across the interface is achieved. By carefully quantifying, experimentally and theoretically, the Fermi level upshift, we show for the first time that this effect is comparable in energy to different plasmonic effects such as the plasmoelectric effect or hot-carriers production. Moreover, by monitoring in situ the adsorption dynamic of halide ions in different AgNP-molecule systems, we show for the first time that the catalytic role of halide ions in plasmonic nanostructures depends on the surface affinity of halide ions compared to that of the target molecule.

Adduct of Aquacobalamin with Hydrogen Peroxide.

Aquacobalamin binds hydrogen peroxide reversibly to form a cobalt(III) hydroperoxo adduct with a 0.25 mM dissociation constant, as evidenced by UV-vis absorption spectroscopy and corroborated by NMR, Raman spectroscopy, stopped-flow UV-vis measurements, and density functional theory calculations.

Cellular Internalization of Beta-Carotene Loaded Polyelectrolyte Multilayer Capsules by Raman Mapping.

Raman mapping is becoming a very useful tool in investigating cells and cellular components, as well as bioactive molecules intracellularly. In this study, we have encapsulated beta-carotene using a layer-by-layer technique, as a way to enhance its stability and bioavailability. Further, we have used Raman mapping to characterize the as-obtained capsules and monitor their uptake by the human retinal epithelial D407 cells. We were able to successfully map the beta-carotene distribution inside the capsules, to localize the capsules intracellularly, and distinguish between capsules and other cellular components.

SERS-based liquid biopsy of saliva and serum from patients with Sjögren's syndrome.

In this preliminary study, we employed surface-enhanced Raman scattering (SERS) of saliva and serum samples for diagnosing Sjogren's syndrome (SjS), a systemic autoimmune disease characterized by dryness of the mouth and eyes. The saliva and serum samples from n = 29 patients with SjS and n = 21 controls were deproteinized with methanol and then the SERS spectra were acquired using silver nanoparticles synthesized by reduction with hydroxylamine hydrochloride. In the case of both saliva and serum, the SERS spectra were dominated by similar bands attributed to purine metabolites such as uric acid, xanthine, and hypoxanthine. Principal component analysis-linear discriminant analysis (PCA-LDA) models built from SERS spectra of saliva and serum yielded an overall classification accuracy of 94% and 98%, respectively. These results suggest that the SERS analysis of saliva and serum is able to capture the complex biochemical perturbations that accompany the onset of SjS, a strategy which could be translated in the future into a novel point-of-care diagnosis method. Graphical abstract.

SERS assessment of the cancer-specific methylation pattern of genomic DNA: towards the detection of acute myeloid leukemia in patients undergoing hematopoietic stem cell transplantation.

In this label-free surface-enhanced Raman scattering (SERS) study of genomic DNA, we demonstrate that the cancer-specific DNA methylation pattern translates into specific spectral differences. Thus, DNA extracted from an acute myeloid leukemia (AML) cell line presented a decreased intensity of the 1005 cm-1 band of 5-methylcytosine compared to normal DNA, in line with the well-described hypomethylation of cancer DNA. The unique methylation pattern of cancer DNA also influences the DNA adsorption geometry, resulting in higher adenine SERS intensities for cancer DNA. The possibility of detecting cancer DNA based on its SERS spectrum was validated on peripheral blood genomic DNA samples from n = 17 AML patients and n = 17 control samples, yielding an overall classification of 82% based on the 1005 cm-1 band of 5-methylcytosine. By demonstrating the potential of SERS in assessing the methylation status in the case of real-life DNA samples, the study paves the way for novel methods of diagnosing cancer. Graphical abstract.

Knee osteoarthritis grading by resonant Raman and surface-enhanced Raman scattering (SERS) analysis of synovial fluid.

In this preliminary study on synovial fluid (SF), knee osteoarthritis (OA) grading of n = 23 patients was accomplished by combining two methods: resonant Raman spectroscopy, and surface-enhanced Raman scattering (SERS) of native proteins acquired with iodide-modified silver nanoparticles and a laser emitting at 633 nm. Based on principal component analysis-linear discriminant analysis (PCA-LDA), the SERS spectra of proteins enabled the classification of low-grade and high-grade OA groups with an accuracy of 91%. Resonant Raman spectra of SF, recorded with laser excitation at 532 nm, exhibited carotenoid-associated bands that were less intense in the case of high-grade knee OA patients. Based on the resonant Raman spectra, the grading of OA patients was accomplished with an accuracy of 74%. Concatenating SERS and Raman spectral information increased the classification accuracy between the two groups to 100%. These results demonstrate the potential of Raman and SERS as a point-of-care method for aiding OA grading.

Raman spectroscopy applications in rheumatology.

Raman spectroscopy is a type of vibrational spectroscopy based on the inelastic scattering of photons, which has attracted much attention due to its potential clinical application in rheumatology. In this review, we discuss the typical spectral features of cartilage, bone, synovial fluid, and pathologic crystal deposits, as well as methods of amplifying the Raman signal of biofluids such as drop-coating deposition Raman spectroscopy. Further, applications of Raman and drop-coating deposition Raman spectroscopy in osteoarthritis are described, highlighting the clinical potential of these methods. We also discuss the role of Raman and related techniques in analyzing pathologic crystals such as monosodium urate, calcium pyrophosphate dihydrate, and hydroxyapatite. The results presented in this review demonstrate that Raman spectroscopy has grown past the stage of proof-of-concept, especially in the case of pathologies involving crystal depositions such as gout and calcium pyrophosphate deposition disease , for which the method has been validated on large number of samples. As the medical community becomes more and more aware of Raman spectroscopy, it is envisioned that it will become a standard technique in the near future.

Warfarin-Capped Gold Nanoparticles: Synthesis, Cytotoxicity, and Cellular Uptake.

Currently, research studies on nanoparticle cytotoxicity, uptake or internalization into the body's cells are of great interest for the improvement of diagnostic and therapeutic applications. We report here the synthesis and characterization of very stable novel warfarin-capped gold nanoparticles with an average diameter of 54 ± 10 nm which were prepared using sodium warfarin as a reducing agent. The nanoparticles were tested in terms of cytotoxicity and cellular internalization in vitro on two cell lines: normal lung fibroblast HFL-1 and human retinal pigment epithelial D407 cells. Our results showed that the normal lung fibroblast HFL-1 cells were more sensitive to the nanoparticle treatment compared to the human retinal pigment epithelial D407 cells. Moreover, any signs of potential cytotoxicity occurred during the first 24 h of treatment, the cellular viability remaining largely unchanged for longer exposure times. Transmission electron microscopy and dark field hyperspectral imaging revealed that the nanoparticles were effectively delivered and released to the HFL-1 and D407 cells' cytoplasm. Our results provide valuable information to further investigate sodium warfarin-capped gold nanoparticles for possible biological applications.

Exosome-carried microRNA-based signature as a cellular trigger for the evolution of chronic lymphocytic leukemia into Richter syndrome.

Even if considered a cumulative and not a proliferative CD5+ B-cell neoplasm, chronic lymphocytic leukemia (CLL) has a proliferation rate higher than that recognized earlier, especially in the lymphoid tissues. Some patients with CLL develop a clinical syndrome entitled Richter syndrome (RS). Understanding CLL genetics and epigenetics may help to elucidate the molecular basics of the clinical heterogeneity of this type of malignancy. In the present project we aimed to identify a microRNA species that can predict the evolution of therapy-resistant CLL towards RS. In the first phase of our study, microRNA-19b was identified as a possible target, and in the second phase, we transfected three different CLL cell lines with microRNA-19b mimic and inhibitor and assessed the potential role on leukemia cells in vitro. The mechanism by which miR-19b acts were identified as the upregulation of Ki67 and downregulation of p53. This was further supported through RT-PCR and western blotting on CLL cell lines, as well as by next generation sequencing on two patients diagnosed with CLL that evolved into RS.