Publications

Abstract The ability to assess molecular binding kinetics in real time is critical for advancing our understanding of molecular interactions in biochemical and biotechnological systems. This work presents a novel optical tweezer (OT)-based method to monitor molecular affinity in real time, focusing on the high-affinity streptavidin-biotin system as a model. Transparent poly(methyl methacrylate) (PMMA) microparticles functionalized with streptavidin were trapped before, during, and after binding with biotinylated bovine serum albumin (biotin-BSA), enabling the analysis of forward-scattered signals to detect nanoscale changes in particle size. By applying the Power Spectral Density method, the friction coefficient of individual particles was calculated, allowing for real-time tracking of binding dynamics and the estimation of the association rate constant (kon approximate to 106M-1s-1). These results are consistent with literature values and demonstrate the potential of this OT-based approach for non-invasive, label-free detection of molecular interactions. Compared to existing techniques, such as atomic force microscopy and cantilever-based sensors, this method offers significant advantages, including real-time monitoring, adaptability to different bioaffinity systems, and compatibility with miniaturized setups. This work establishes a foundation for using OT-based tools to monitor high-affinity molecular interactions in real time. While demonstrated here using biotinylated BSA as a model ligand, future studies will explore the method's applicability to smaller ligands and more subtle surface modifications.

Abstract The development of simple, selective, and cost-effective methods for quantification of bovine serum albumin (BSA) is currently very important for assessing milk quality (and safety). In this work, a new surface plasmon resonance (SPR) sensor was developed, consisting of imprinted hydrogel-based nanoparticles (nanoMIPs) immobilized on gold platforms, to quantify BSA in bovine milk. The nanoMIPs prepared for recognition of BSA were synthesized by the precipitation polymerization approach, using a synthetic BSA epitope (VVSTQTALA) as template. The spherical MIP nanoparticles (NPs) had an average size of 60 nm. The binding studies performed revealed that the binding affinity of the prepared nanoMIPs to BSA (KD = 7.1 × 10−6 mol L−1) was comparable to that obtained by a natural BSA antibody (KD = 2.5 × 10−6 mol L−1). The plasmonic sensor incorporating the MIP nanomaterials achieved a limit of detection (LOD) of 1.02 × 10−6 mol L−1 (0.068 mg mL−1) and a limit of quantification (LOQ) of 3.39 × 10−6 mol L−1 (0.225 mg mL−1), over a linear range from 2.0 × 10−6 mol L−1 to 1.5 × 10−5 mol L−1. Moreover, the selectivity studies revealed a significant sensor response towards casein and a negligible response towards vancomycin. In the end, the optical sensor was tested against commercial milk samples, showing promising viability for detection of BSA as the value reported by the plasmonic sensor ((1.0 ± 0.1) × 10−4 mol L−1) was very close to that obtained by size exclusion-high-performance liquid chromatography (SEC-HPLC). © 2025 The Author(s)

Abstract Hydroponics is an advanced agricultural technique that involves growing plants without soil. Instead, plants are cultivated in a nutrient-rich water solution that provides all the essential minerals they need to thrive, allowing plants to grow either with their roots directly in the solution or supported by inert substrates like pine bark, coconut husk fiber, and rice husk. The solid waste generated from hydroponic cultivation is valuable due to its low cost, abundance, biodegradability, and renewability. These residues are rich in lignocellulosic materials, which can be extracted and refined to produce cellulose and nanocellulose (NC). In this work, cellulose and nanocellulose were extracted from residues of coconut husk fiber and a mixture of pine bark and coconut husk fiber, used in tomato and strawberry hydroponics, respectively. The residues were ground, washed, and chemically treated to obtain cellulose and NC. The chemical process involved several stages: (i) acid treatment, alkaline treatment, and bleaching to isolate cellulose, and (ii) acid hydrolysis followed by ultrasonication to obtain NC. Both materials underwent characterization using various techniques such as TGA, DSC, XRD and FTIR-ATR, which confirmed very low levels of lignin and hemicellulose. Morphological characterization through SEM revealed the presence of micro- and nano-crystals in the cellulose and NC samples, respectively, highlighting the effectiveness of the extraction method. The high purity and quality of the extracted materials make them competitive with commercially available products, suitable for applications in healthcare, food packaging, and automotive industries, while supporting recycling and reuse principles.

Abstract Active and abandoned mining sites are significant sources of heavy metals and metalloid pollution, leading to serious environmental issues. This study assessed the environmental risks posed by potentially toxic elements (PTEs), specifically arsenic (As) and antimony (Sb), in the Technosols (mining residues) of the former Pej & atilde;o coal mine complex in Northern Portugal, a site impacted by forest wildfires in October 2017 that triggered underground combustion within the waste heaps. Our methodology involved determining the pseudo-total concentrations of As and Sb in the collected heap samples using microwave digestion with aqua regia (ISO 12914), followed by analysis using hydride generation-atomic absorption spectroscopy (HG-AAS). The concentrations of As an Sb ranging from 31.0 to 68.6 mg kg-1 and 4.8 to 8.3 mg kg-1, respectively, were found to be above the European background values reported in project FOREGS (11.6 mg kg-1 for As and 1.04 mg kg-1 for Sb) and Portuguese Environment Agency (APA) reference values for agricultural soils (11 mg kg-1 for As and 7.5 mg kg-1 for Sb), indicating significant enrichment of these PTEs. Based on average Igeo values, As contamination overall was classified as unpolluted to moderately polluted while Sb contamination was classified as moderately polluted in the waste pile samples and unpolluted to moderately polluted in the downhill soil samples. However, total PTE content alone is insufficient for a comprehensive environmental risk assessment. Therefore, further studies on As and Sb fractionation and speciation were conducted using the Shiowatana sequential extraction procedure (SEP). The results showed that As and Sb levels in the more mobile fractions were not significant. This suggests that the enrichment in the burned (BCW) and unburned (UCW) coal waste areas of the mine is likely due to the stockpiling of lithic fragments, primarily coals hosting arsenian pyrites and stibnite which largely traps these elements within its crystalline structure. The observed enrichment in downhill soils (DS) is attributed to mechanical weathering, rock fragment erosion, and transport processes. Given the strong association of these elements with solid phases, the risk of leaching into surface waters and aquifers is considered low. This work underscores the importance of a holistic approach to environmental risk assessment at former mining sites, contributing to the development of sustainable remediation strategies for long-term environmental protection.

Abstract This paper describes the development of an optical tweezers system that operates in fully automatic mode. It features image recognition for particle tracking, allowing for the optical trapping and analysis of identified targets. The system can perform analysis of forward scattered light and Raman spectroscopy of the trapped particles, facilitating the automated analysis of a large number of samples without manual intervention. By leveraging combined analytical methods and AI for robust classification, this system contributes to the advancement of automated diagnostic tools. Preliminary results demonstrate the system's effectiveness using different kinds of standard and biofunctionalized PMMA microparticles.

Abstract Purpose Pejao Mining Complex locates in Castelo de Paiva municipality and, until its closure in 1994, was one of the most important coal mines in the Douro Coalfield. This work aims to study the presence, quantify, and evaluate the dissemination of mercury (Hg), a potentially toxic element (PTE) of major public health concern by the World Health Organization (WHO), from a waste pile affected by coal fires.Materials and methods Samples from areas affected and unaffected by the combustion and from surrounding soil were collected from Fojo waste pile region. First, the Hg pseudo-total concentration was estimated for all collected samples by soil microwave-assisted digestion with aqua regia (USEPA 3051A). Then, a sequential extraction procedure (SEP), the USEPA 3200, was applied for Hg fractionation and speciation aiming to evaluate Hg mobility and bioavailability to surrounding ecosystems.Results and discussion The results obtained showed a Hg enrichment in soil samples when compared to Portuguese and international reference values for soils. Relatively to the Hg availability and mobility, although it predominates in the semi-mobile fraction, the waste pile materials exposed to combustion showed a concerning increase of Hg levels in the mobile fraction that contains the more labile Hg species, being a major source of environmental contamination by Hg.Conclusions This study allowed to conclude that combustion of mining residues increased Hg mobility, toxicity, and bioavailability, increasing the contamination potential of the coal waste pile. The methodology applied in this work can be replicated in other abandoned mines to monitor, control, and/or mitigate the Hg environmental impact in the surrounding soils and waters.

Abstract New systems with innovative design to perform measurements combining electrochemistry and surface plasmon resonance (ESPR) are currently a need to overcome the limitations of existent market solutions and expand the research possibilities of this technology. The main goal of this work was to develop a new cell to increase ESPR practical applications in several fields. To do so, a homemade SPR cell, fabricated by 3D-printing technology, was adapted for this purpose by incorporating the conventional 3-electrodes to perform the electrochemical experiments. The developed cell was fully compatible with commercial SPR substrates. After optimization of the homemade ESPR setup to perform the combined electrochemical and SPR measurements, two main applications were explored in this work. The first was the use of ESPR technology as straightforward tool to simultaneously investigate the electrical and optical properties of conducing/nonconducting polymers electrosynthetized on the SPR platforms. The conducting polymer poly(thionine) was used in this work for proof-of- concept. The second application envisaged the use of ESPR approach for simple electrodeposition of materials with enhanced plasmonic properties for sensitivity enhancement of SPR biosensors. For validation of the concept, graphene oxide (GO) was electrochemically reduced on gold substrates aiming to evaluate the plasmonic properties of graphene-modified sensing surfaces.

Abstract The field of electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) has been continuously expanding over the years due to their vast number of applications, including to investigate the partitioning of ionizable drugs at liquid-liquid systems. The aim of this Review is to highlight the great potential of ITIES as simple model of biological membranes to gather information on drug partition, lipophilicity, and pharmacokinetics that can be very useful for researchers in the field of drug discovery for development of new drugs with enhanced permeability. Relevant contributions and perspectives to improve the applicability of ITIES in partition studies were highlighted and discussed. The second part of this Review pretends to highlight the application of electrochemistry at the ITIES as experimental technique to investigate interactions between small ligands, including drugs, and DNA, a topic of high research interest in pharmaceutical and biological sciences, which remains with lots of opportunities to explore. Voltammetry at Liquid-Liquid Interfaces can be a versatile tool in the field of Drug Discovery as simple model for mimicking drug permeation through biological membranes helping to understand the partition of ionizable drugs between the aqueous and organic phases while providing fundamental information on its lipophilicity that can contribute to the design of new drugs with improved biological activity. image

Abstract Electrochemical impedance spectroscopy (EIS) is a reliable technique for gathering information about electrochemical process occurring at the electrode surface and investigating properties of materials. Furthermore, EIS technique can be a very versatile and valuable tool in analytical assays for detection and quantification of several chemically and biologically relevant (bio)molecules. The first part of this Review (Introduction) provides brief insights into (i) theoretical aspects of EIS, (ii) the instrumentation required to perform the EIS studies and (iii) the most relevant representations of impedance experimental data (such as Nyquist and Bode plots). In the end of this section, (iv) theoretical aspects regarding the fitting of the Randles circuit to experimental data are addressed, not only to obtain information about electrochemical processes but also to illustrate its utility for analytical purposes. The second part of the Review (Impedimetric Detection of Disease Biomarkers) focuses on the applications of EIS in the biomedical field, particularly as analytical technique in electrochemical sensors and biosensors for screening disease biomarkers. In the last section (Conclusions and Perspectives), we discuss main achievements of EIS technique in analytical assays and provide some perspectives, challenges and future applications in the biomedical field.