Label-free SERS assay combined with multivariate spectral data analysis for lamotrigine quantification in human serum.

Considering the need for a more time and cost-effective method for lamotrigine (LTG) detection in clinics we developed a fast and robust label-free assay based on surface-enhanced Raman scattering (SERS) for LTG quantification from human serum. The optimization and application of the developed assay is presented  showing the: (i) exploration of different methods for LTG separation from human serum; (ii) implementation of a molecular adsorption step on an ordered Au nanopillar SERS substrate; (iii) adaptation of a fast scanning of the SERS substrate, performed with a custom-built compact Raman spectrometer; and (iv) development of LTG quantification methods with univariate and multivariate spectral data analysis. Our results showed, for the first time, the SERS-based characterization of LTG and its label-free identification in human serum. We found that combining a miniaturized solid phase extraction, as sample pre-treatment with the SERS assay, and using a multivariate model is an optimal strategy for LTG quantification in human serum in a linear range from 9.5 to 75 µM, with LoD and LoQ of 3.2 µM and 9.5 µM, respectively, covering the suggested clinical therapeutic window. We also showed that the developed assay allowed for quantifying LTG from human serum in the presence of other drugs, thereby demonstrating the robustness of label-free SERS. The sensing approach and instrumentation can be further automated and integrated in devices that can advance the drug monitoring in real clinical settings.

SERS-based detection of 5-S-cysteinyl-dopamine as a novel biomarker of Parkinson's disease in artificial biofluids.

The early detection of Parkinson's disease (PD) can significantly improve treatment and quality of life in patients. 5-S-Cysteinyl-dopamine (CDA) is a key metabolite of high relevance for the early detection of PD. Therefore, its sensitive detection with fast and robust methods can improve its use as a biomarker. In this work we show the potentialities of label-free SERS spectroscopy in detecting CDA in aqueous solutions and artificial biofluids, with a simple, fast and sensitive approach. We present a detailed experimental SERS band assignment of CDA employing silver nanoparticle (AgNP) substrates in aqueous media, which was supported by theoretical calculations and simulated Raman and SERS spectra. The tentative orientation of CDA over the AgNP was also studied, indicating that catechol and carboxylic acid play a key role in the metallic surface adsorption. Moreover, we showed that SERS can allow us to identify CDA in aqueous media at low concentration, leading to the identification of some of its characteristic bands in pure water and in synthetic cerebrospinal fluid (SCSF) below 1 × 10-8 M, while its band identification in simulated urine (SUR) can be reached at 1 × 10-7 M. In conclusion, we show that CDA can be suitably detected by means of label-free SERS spectroscopy, which can significantly improve its sensitive detection for further analytical studies as a novel biomarker and further clinical diagnosis in PD patients.

Graphene-Based Biosensors for Molecular Chronic Inflammatory Disease Biomarker Detection.

Chronic inflammatory diseases, such as cancer, diabetes mellitus, stroke, ischemic heart diseases, neurodegenerative conditions, and COVID-19 have had a high number of deaths worldwide in recent years. The accurate detection of the biomarkers for chronic inflammatory diseases can significantly improve diagnosis, as well as therapy and clinical care in patients. Graphene derivative materials (GDMs), such as pristine graphene (G), graphene oxide (GO), and reduced graphene oxide (rGO), have shown tremendous benefits for biosensing and in the development of novel biosensor devices. GDMs exhibit excellent chemical, electrical and mechanical properties, good biocompatibility, and the facility of surface modification for biomolecular recognition, opening new opportunities for simple, accurate, and sensitive detection of biomarkers. This review shows the recent advances, properties, and potentialities of GDMs for developing robust biosensors. We show the main electrochemical and optical-sensing methods based on GDMs, as well as their design and manufacture in order to integrate them into robust, wearable, remote, and smart biosensors devices. We also describe the current application of such methods and technologies for the biosensing of chronic disease biomarkers. We also describe the current application of such methods and technologies for the biosensing of chronic disease biomarkers with improved sensitivity, reaching limits of detection from the nano to atto range concentration.

Carbon SH-SAW-Based Electronic Nose to Discriminate and Classify Sub-ppm NO2.

In this research, a compact electronic nose (e-nose) based on a shear horizontal surface acoustic wave (SH-SAW) sensor array is proposed for the NO2 detection, classification and discrimination among some of the most relevant surrounding toxic chemicals, such as carbon monoxide (CO), ammonia (NH3), benzene (C6H6) and acetone (C3H6O). Carbon-based nanostructured materials (CBNm), such as mesoporous carbon (MC), reduced graphene oxide (rGO), graphene oxide (GO) and polydopamine/reduced graphene oxide (PDA/rGO) are deposited as a sensitive layer with controlled spray and Langmuir-Blodgett techniques. We show the potential of the mass loading and elastic effects of the CBNm to enhance the detection, the classification and the discrimination of NO2 among different gases by using Machine Learning (ML) techniques (e.g., PCA, LDA and KNN). The small dimensions and low cost make this analytical system a promising candidate for the on-site discrimination of sub-ppm NO2.

Three-Dimensional Porous Scaffolds Derived from Bovine Cancellous Bone Matrix Promote Osteoinduction, Osteoconduction, and Osteogenesis.

The use of three-dimensional porous scaffolds derived from decellularized extracellular matrix (ECM) is increasing for functional repair and regeneration of injured bone tissue. Because these scaffolds retain their native structures and bioactive molecules, in addition to showing low immunogenicity and good biodegradability, they can promote tissue repair and regeneration. Nonetheless, imitating these features in synthetic materials represents a challenging task. Furthermore, due to the complexity of bone tissue, different processes are necessary to maintain these characteristics. We present a novel approach using decellularized ECM material derived from bovine cancellous bone by demineralization, decellularization, and hydrolysis of collagen to obtain a three-dimensional porous scaffold. This study demonstrates that the three-dimensional porous scaffold obtained from bovine bone retained its osteoconductive and osteoinductive properties and presented osteogenic potential when seeded with human Wharton's jelly mesenchymal stromal cells (hWJ-MSCs). Based on its characteristics, the scaffold described in this work potentially represents a therapeutic strategy for bone repair.

SERS characterization of dopamine and in situ dopamine polymerization on silver nanoparticles.

Dopamine (DA) regulates several functions in the central nervous system and its depletion is responsible for psychological disorders like Parkinson's disease. Several analytical approaches have been presented for DA detection in pathological diagnosis. SERS spectroscopy is a highly promising technique for the sensitive detection of DA. However, an improvement in its detection in aqueous solution is highly desirable for reliable quantification in biological fluids. In this work, we explored a label-free SERS approach for DA detection, employing two conventional methods to synthesize Ag colloids: reduction via citrates (c-AgNPs) and reduction via hydroxylamine (h-AgNPs), and SERS measurements were performed with a laser at 488 nm wavelength. Under these conditions, DA was identified through reproducible SERS spectra in the c-AgNP medium; however, the SERS spectra of DA in h-AgNP solution showed a completely different SERS profile. SERS band analysis revealed that DA in h-AgNPs was oxidized and converted into polydopamine (PDA), which was triggered after exposure to laser radiation. DA oxidation and PDA formation were followed over time through the SERS band profile at pH 7, 9 and 12. We found that in situ PDA formation started after 50 min of laser irradiation of DA at pH 7, while DA was quickly oxidized at pH 9 and 12. Here, we present a detailed SERS band analysis of PDA, which sheds light on the molecular steps in the pathway formation of the PDA structure. Spectroscopic analysis and characterization revealed that a long laser exposure time led to the formation of stable PDA complexes with AgNPs, which allowed us to propose a novel approach for synthesis of AgNP-PDA composites. In conclusion, to detect DA through a label-free SERS approach, c-AgNPs must be employed, while stable AgNP-PDA materials can be achieved with h-AgNPs and 488 nm laser excitation.

Graphenic substrates as modifiers of the emission and vibrational responses of interacting molecules: The case of BODIPY dyes.

Graphenic substrates (GS), such as reduced graphene oxide (rGO) and graphene oxide (GO), are 2D materials known for their unique physicochemical properties such as their ability to enhance vibrational spectroscopic signals and quench the fluorescence of adsorbed molecules. These properties provide an opportunity to develop nanostructured GS-based systems for detecting and identifying different analytes with high sensitivity and reliability through molecular spectroscopic techniques. This work evaluated the capacities of different GS to interact with a highly fluorescent compound, thereby changing its optical emission response (fluorescence quenching) and amplifying its vibrational signal, which is the base of graphene-enhanced Raman scattering (GERS). To test these properties, we used a derivative of highly fluorescent BODIPY (BP) compounds, which cover a wide range of applications from solar energy conversion to photodynamic cancer therapy. GS prepared by using the Langmuir-Blodgett (LB) technique allowed us to quench the fluorescence emission of BP and improve its Raman spectroscopy detection limit due to the GERS effect. These results were interpreted in light of the π-π interactions taking place between the Csp2 domains of GS and the aromatic core of the BP fluorophore.

The Peptide AmPep1 Derived from Amaranth Recognizes the Replication Hairpin of TYLCV Disturbing Its Replication Process in Host Plants.

Antiviral compounds targeting viral replicative processes have been studied as an alternative for the control of begomoviruses. Previously, we have reported that the peptide AmPep1 has strong affinity binding to the replication origin sequence of tomato yellow leaf curl virus (TYLCV). In this study, we describe the mechanism of action of this peptide as a novel alternative for control of plant-infecting DNA viruses. When AmPep1 was applied exogenously to tomato and Nicotiana benthamiana plants infected with TYLCV, a decrease in the synthesis of the two viral DNA strands (CS and VS) was observed, with a consequent delay in the development of disease progress in treated plants. The chemical mechanism of action of AmPep1 was deduced using Raman spectroscopy and molecular modeling showing the formation of chemical interactions such as H bonds and electrostatic interactions and the formation of π-π interactions between both biomolecules contributing to tampering with the viral replication.

5-S-cysteinyl-dopamine, a neurotoxic endogenous metabolite of dopamine: Implications for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and is characterized for being an idiopathic and multifactorial disease. Extensive research has been conducted to explain the origin of the disease, but it still remains elusive. It is well known that dopamine oxidation, through the endogenous formation of toxic metabolites, is a key process in the activation of a cascade of molecular events that leads to cellular death in the hallmark of PD. Thio-catecholamines, such as 5-S-cysteinyl-dopamine, 5-S-glutathionyl-dopamine and derived benzothiazines, are endogenous metabolites formed in the dopamine oxidative degradation pathway. Those metabolites have been shown to be highly toxic to neurons in the substantia nigra pars compacta, activating molecular mechanisms that ultimately lead to neuronal death. In this review we describe the origin, formation and the toxic effects of 5-S-cysteinyl-dopamine and its oxidative derivatives that cause death to dopaminergic neurons. Furthermore, we correlate the formation of those metabolites with the neurodegeneration progress in PD. In addition, we present the reported neuroprotective strategies of products that protect against the cellular damage of those thio-catecholamines. Finally, we discuss the advantages in the use of 5-S-cysteinyl-dopamine as a potential biomarker for PD.

Sensitive Raman detection of human recombinant interleukin-6 mediated by DCDR/GERS hybrid platforms.

Recombinant human interleukin-6 (IL-6) is a key cytokine that plays an important role in the immune system and inflammatory response, explaining why any modification of its concentration in biological fluids is considered a signal of a pathological condition. Therefore, it is important to develop alternative, highly sensitive and reliable analytical methodologies to detect and identify this analyte in biological fluids. Herein, we present a proof of concept for the development of a new analytical hybrid platform for IL-6 detection that is based on the combination of drop-coating deposition Raman (DCDR) spectroscopy and graphene-enhanced Raman spectroscopy (GERS) effects. The sensitivity limits for IL-6 detection were found to be a function of the type of substrate used. When a 1 µL droplet of IL-6 solution is deposited and dried on an Si substrate, a DCDR effect occurs, and a detection limit below 1 ng mL-1 is obtained; however, when the same is performed using a hybrid substrate of reduced graphene oxide and silicon (rGO/Si), the joint action of DCDR and GERS effects results in a detection limit well below 1 pg mL-1. It is important to note that this result implies the absolute mass detection of 1 fg of IL-6. In summary, the Raman spectroscopy DCDR/GERS analytical platform proposed here allows the reliable identification of, as well as the very sensitive detection of, IL-6 and promises to improve the performance of clinical evaluations of this biomarker that are currently in use. In this study, the Raman spectra of IL-6 in powder and solution, together with the corresponding band assignment, are presented for the first time in the literature.

Structural Changes of Amyloid Beta in Hippocampus of Rats Exposed to Ozone: A Raman Spectroscopy Study.

The aim of this work was to study the effect of oxidative stress on the structural changes of the secondary peptide structure of amyloid beta 1-42 (Aß 1-42), in the dentate gyrus of hippocampus of rats exposed to low doses of ozone. The animals were exposed to ozone-free air (control group) and 0.25 ppm ozone during 7, 15, 30, 60, and 90 days, respectively. The samples were studied by: (1) Raman spectroscopy to detect the global conformational changes in peptides with α-helix and ß-sheet secondary structure, following the deconvolution profile of the amide I band; and (2) immunohistochemistry against Aß 1-42. The results of the deconvolutions of the amide I band indicate that, ozone exposure causes a progressively decrease in the abundance percentage of α-helix secondary structure. Furthermore, the ß-sheet secondary structure increases its abundance percentage. After 60 days of ozone exposure, the ß-sheet band is identified in a similar wavenumber of the Aß 1-42 peptide standard. Immunohistochemistry assays show an increase of Aß 1-42 immunoreactivity, coinciding with the conformational changes observed in the Raman spectroscopy of Aß 1-42 at 60 and 90 days. In conclusion, oxidative stress produces changes in the folding process of amyloid beta peptide structure in the dentate gyrus, leading to its conformational change in a final ß-sheet structure. This is associated to an increase in Aß 1-42 expression, similar to the one that happens in the brain of Alzheimer's Disease (AD) patients.