An ultrasensitive paper-based SERS sensor for detection of nucleolin using silver-nanostars, plastic antibodies and natural antibodies.

A state-of-the-art, ultrasensitive, paper-based SERS sensor has been developed using silver nanostars (AgNSs) in combination with synthetic and natural antibodies. A key component of this innovative sensor is the plastic antibody, which was synthesized using molecularly imprinted polymer (MIP) technology. This ground-breaking combination of paper substrates/MIPs with AgNSs, which is similar to a sandwich immunoassay, is used for the first time with the aim of SERS detection and specifically targets nucleolin (NCL), a cancer biomarker. The sensor device was carefully fabricated by synthesizing a polyacrylamide-based MIP on cellulose paper (Whatman Grade 1 filter) by photopolymerization. The binding of NCL to the MIP was then confirmed by natural antibody binding using a sandwich assay for quantitative SERS analysis. To facilitate the detection of NCL, antibodies were pre-bound to AgNSs with a Raman tag so that the SERS signal could indicate the presence of NCL. The composition of the sensory layers/materials was meticulously optimized. The intensity of the Raman signal at ∼1078 cm-1 showed a linear trend that correlated with increasing concentrations of NCL, ranging from 0.1 to 1000 nmol L-1, with a limit of detection down to 0.068 nmol L-1 in human serum. The selectivity of the sensor was confirmed by testing its analytical response in the presence of cystatin C and lysozyme. The paper-based SERS detection system for NCL is characterized by its simplicity, sustainability, high sensitivity and stability and thus embodies essential properties for point-of-care applications. This approach is promising for expansion to other biomarkers in various fields, depending on the availability of synthetic and natural antibodies.

Artificial Dendritic Cells: A New Era of Promising Antitumor Immunotherapy.

The accelerated development of antitumor immunotherapies in recent years has brought immunomodulation into the spotlight. These include immunotherapeutic treatments with dendritic cell (DC)-based vaccines which can elicit tumor-specific immune responses and prolong survival. However, this personalized treatment has several drawbacks, including being costly, labor-intensive, and time consuming. This has sparked interest in producing artificial dendritic cells (aDCs) to open up the possibility of standardized "off-the-shelf" protocols and circumvent the cumbersome and expensive personalized medicine. aDCs take advantage of materials that can be designed and tailored for specific clinical applications. Here, an overview of the immunobiology underlying antigen presentation by DCs is provided in an attempt to select the key features to be mimicked and/or improved through the development of aDCs. The inherent properties of aDCs that greatly impact their performance in vivo and, consequently, the fate of the triggered immune response are also outlined.

An ultra-sensitive electrochemical biosensor using the Spike protein for capturing antibodies against SARS-CoV-2 in point-of-care.

This work presents an innovative ultra-sensitive biosensor having the Spike protein on carbon-based screen-printed electrodes (SPEs), for monitoring in point-of-care antibodies against SARS-CoV-2, a very important tool for epidemiological monitoring of COVID-19 infection and establishing vaccination schemes. In an innovative and simple approach, a highly conductive support is combined with the direct adsorption of Spike protein to enable an extensive antibody capture. The high conductivity was ensured by using carboxylated carbon nanotubes on the carbon electrode, by means of a simple and quick approach, which also increased the surface area. These were then modified with EDC/NHS chemistry to produce an amine layer and undergo Spike protein adsorption, to generate a stable layer capable of capturing the antibodies against SARS-CoV-2 in serum with great sensitivity. Electrochemical impedance spectroscopy was used to evaluate the analytical performance of this biosensor in serum. It displayed a linear response between 1.0 â€‹pg/mL and 10 â€‹ng/mL, with a detection limit of ∼0.7 â€‹pg/mL. The analysis of human positive sera containing antibody in a wide range of concentrations yielded accurate data, correlating well with the reference method. It also offered the unique ability of discriminating antibody concentrations in sera below 2.3 â€‹µg/mL, the lowest value detected by the commercial method. In addition, a proof-of-concept study was performed by labelling anti-IgG antibodies with quantum dots to explore a new electrochemical readout based on the signal generated upon binding to the anti-S protein antibodies recognised on the surface of the biosensor. Overall, the alternative serologic assay presented is a promising tool for assessing protective immunity to SARS-CoV-2 and a potential guide for revaccination.

A molecularly imprinted photonic polymer based on an inverse opal structure for sensing D-dimer at the point-of-care.

Accurate and timely diagnosis of venous thromboembolism (VTE) is crucial to prevent related morbidity and mortality. This work reports a label-free sensor for D-dimer, a biomarker of VTE. The sensor is based on the synergy between the colloidal crystal templating method and the molecular imprinting technique. The design of the photonic molecularly imprinted polymer (PMIP) is focused on the preparation of an inverse opal structure, resulting from silica infiltration in a poly(methyl methacrylate) photonic crystal template, followed by a calcination stage that removes the sacrificial colloidal crystal. The molecularly imprinted polymer in the inverse opal structure is then synthesized in the presence of the template molecule, the peptide D-dimer. After D-Dimer removal, the PMIP consists in a three-dimensional highly ordered structure, where nanocavities complementary to the D-dimer in shape and binding features are distributed. The PMIP showed a linear response to D-dimer in synthetic urine, exhibiting a decrease in the reflectance intensity with increasing D-dimer concentrations, ranging from 22.5 ng mL-1 to 1450.0 ng mL-1. The PMIP material demonstrated a limit of detection of 15.5 ng mL-1 and was selective for D-dimer in the presence of fibrinopeptide B, another prospective VTE biomarker in urine. Moreover, the sensor was reusable up to five times without losing its recognition ability. Overall, a novel PMIP material is described for successful recognition of D-Dimer. Considering the clinical relevance of D-dimer detection, the sensor is envisioned as a promising low-cost test for urinalysis.

Emerging Optical Materials in Sensing and Discovery of Bioactive Compounds.

Optical biosensors are used in numerous applications and analytical fields. Advances in these sensor platforms offer high sensitivity, selectivity, miniaturization, and real-time analysis, among many other advantages. Research into bioactive natural products serves both to protect against potentially dangerous toxic compounds and to promote pharmacological innovation in drug discovery, as these compounds have unique chemical compositions that may be characterized by greater safety and efficacy. However, conventional methods for detecting these biomolecules have drawbacks, as they are time-consuming and expensive. As an alternative, optical biosensors offer a faster, simpler, and less expensive means of detecting various biomolecules of clinical interest. In this review, an overview of recent developments in optical biosensors for the detection and monitoring of aquatic biotoxins to prevent public health risks is first provided. In addition, the advantages and applicability of these biosensors in the field of drug discovery, including high-throughput screening, are discussed. The contribution of the investigated technological advances in the timely and sensitive detection of biotoxins while deciphering the pathways to discover bioactive compounds with great health-promoting prospects is envisaged to meet the increasing demands of healthcare systems.

Biosensors for European Zoonotic Agents: A Current Portuguese Perspective.

Emerging and recurrent outbreaks caused by zoonotic agents pose a public health risk. They result in morbidity and mortality in humans and significant losses in the livestock and food industries. This highlights the need for rapid surveillance methods. Despite the high reliability of conventional pathogen detection methods, they have high detection limits and are time-consuming and not suitable for on-site analysis. Furthermore, the unpredictable spread of zoonotic infections due to a complex combination of risk factors urges the development of innovative technologies to overcome current limitations in early warning and detection. Biosensing, in particular, is highlighted here, as it offers rapid and cost-effective devices for use at the site of infection while increasing the sensitivity of detection. Portuguese research in biosensors for zoonotic pathogens is the focus of this review. This branch of research produces exciting and innovative devices for the study of the most widespread pathogenic bacteria. The studies presented here relate to the different classes of pathogens whose characteristics and routes of infection are also described. Many advances have been made in recent years, and Portuguese research teams have increased publications in this field. However, biosensing still needs to be extended to other pathogens, including potentially pandemic viruses. In addition, the use of biosensors as part of routine diagnostics in hospitals for humans, in animal infections for veterinary medicine, and food control has not yet been achieved. Therefore, a convergence of Portuguese efforts with global studies on biosensors to control emerging zoonotic diseases is foreseen for the future.

Molecular Imprinting on Nanozymes for Sensing Applications.

As part of the biomimetic enzyme field, nanomaterial-based artificial enzymes, or nanozymes, have been recognized as highly stable and low-cost alternatives to their natural counterparts. The discovery of enzyme-like activities in nanomaterials triggered a broad range of designs with various composition, size, and shape. An overview of the properties of nanozymes is given, including some examples of enzyme mimics for multiple biosensing approaches. The limitations of nanozymes regarding lack of selectivity and low catalytic efficiency may be surpassed by their easy surface modification, and it is possible to tune specific properties. From this perspective, molecularly imprinted polymers have been successfully combined with nanozymes as biomimetic receptors conferring selectivity and improving catalytic performance. Compelling works on constructing imprinted polymer layers on nanozymes to achieve enhanced catalytic efficiency and selective recognition, requisites for broad implementation in biosensing devices, are reviewed. Multimodal biomimetic enzyme-like biosensing platforms can offer additional advantages concerning responsiveness to different microenvironments and external stimuli. Ultimately, progress in biomimetic imprinted nanozymes may open new horizons in a wide range of biosensing applications.

Photonics in nature and bioinspired designs: sustainable approaches for a colourful world.

Biological systems possess nanoarchitectures that have evolved for specific purposes and whose ability to modulate the flow of light creates an extraordinary diversity of natural photonic structures. In particular, the striking beauty of the structural colouration observed in nature has inspired technological innovation in many fields. Intense research has been devoted to mimicking the unique vivid colours with newly designed photonic structures presenting stimuli-responsive properties, with remarkable applications in health care, safety and security. This review highlights bioinspired photonic approaches in this context, starting by presenting many appealing examples of structural colours in nature, followed by describing the versatility of fabrication methods and designed coloured structures. A particular focus is given to optical sensing for medical diagnosis, food control and environmental monitoring, which has experienced a significant growth, especially considering the advances in obtaining inexpensive miniaturized systems, more reliability, fast responses, and the use of label-free layouts. Additionally, naturally derived biomaterials and synthetic polymers are versatile and fit many different structural designs that are underlined. Progress in bioinspired photonic polymers and their integration in novel devices is discussed since recent developments have emerged to lift the expectations of smart, flexible, wearable and portable sensors. The discussion is expanded to give emphasis on additional functionalities offered to related biomedical applications and the use of structural colours in new sustainable strategies that could meet the needs of technological development.

Imprinting Technology in Electrochemical Biomimetic Sensors.

Biosensors are a promising tool offering the possibility of low cost and fast analytical screening in point-of-care diagnostics and for on-site detection in the field. Most biosensors in routine use ensure their selectivity/specificity by including natural receptors as biorecognition element. These materials are however too expensive and hard to obtain for every biochemical molecule of interest in environmental and clinical practice. Molecularly imprinted polymers have emerged through time as an alternative to natural antibodies in biosensors. In theory, these materials are stable and robust, presenting much higher capacity to resist to harsher conditions of pH, temperature, pressure or organic solvents. In addition, these synthetic materials are much cheaper than their natural counterparts while offering equivalent affinity and sensitivity in the molecular recognition of the target analyte. Imprinting technology and biosensors have met quite recently, relying mostly on electrochemical detection and enabling a direct reading of different analytes, while promoting significant advances in various fields of use. Thus, this review encompasses such developments and describes a general overview for building promising biomimetic materials as biorecognition elements in electrochemical sensors. It includes different molecular imprinting strategies such as the choice of polymer material, imprinting methodology and assembly on the transduction platform. Their interface with the most recent nanostructured supports acting as standard conductive materials within electrochemical biomimetic sensors is pointed out.

PLGA nanoparticles as a platform for vitamin D-based cancer therapy.

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were studied as drug delivery vehicles for calcitriol, the active form of vitamin D3. In vitro effects of calcitriol encapsulated in PLGA nanoparticles were evaluated with respect to free calcitriol on human pancreatic cell lines, S2-013 and hTERT-HPNE, and the lung cancer cell line A549. Encapsulated calcitriol retained its biological activity, reducing the cell growth. Cytotoxicity assays demonstrated that encapsulation of calcitriol enhanced its inhibitory effect on cell growth at a concentration of 2.4 µM for the S2-013 cells (91%) and for A549 cells (70%) comparared to the free calcitriol results. At this concentration the inhibitory effect on nontumor cells (hTERT-HPNE) decreased to 65%. This study highlights the ability of PLGA nanoparticles to deliver vitamin D3 into cancer cells, with major effects regarding cancer cell cycle arrest and major changes in the cell morphological features.

Transferrin surface-modified PLGA nanoparticles-mediated delivery of a proteasome inhibitor to human pancreatic cancer cells.

The aim of this study was to develop a drug delivery system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles for an efficient and targeted action of the proteasome inhibitor bortezomib against pancreatic cancer cells. The PLGA nanoparticles were formulated with a poloxamer, and further surface-modified with transferrin for tumor targeting. The nanoparticles were characterized as polymer carriers of bortezomib, and the cellular uptake and growth inhibitory effects were evaluated in pancreatic cells. Cellular internalization of nanoparticles was observed in normal and cancer cells, but with higher uptake by cancer cells. The sustained release of the loaded bortezomib from PLGA nanoparticles showed cytotoxic effects against pancreatic normal and cancer cells. Noteworthy differential cytotoxicity was attained by transferrin surface-modified PLGA nanoparticles since significant cell growth inhibition by delivered bortezomib was only observed in cancer cells. These findings demonstrate that the ligand transferrin enhanced the targeted delivery of bortezomib-loaded PLGA nanoparticles to pancreatic cancer cells. These in vitro results highlight the transferrin surface-modified PLGA nanoparticles as a promising system for targeted delivery of anticancer drugs.

Localization and properties of cholinesterases in the common prawn (Palaemon serratus): A kinetic-histochemical study.

Due to the diversity in biochemical properties and tissue distribution of cholinesterase (ChE) enzymes, a characterization should be performed before they are used as biomarkers for monitoring pesticide contamination. In this study we investigate the distribution of ChEs and their kinetic properties in diverse tissues of the common prawn Palaemon serratus. The results concerning the histochemical localization of ChEs suggest that the highest amount of ChE activity occurs in prawn eyes, followed by the brain, gills, and digestive tract. Negligible staining was observed in the muscle. We describe the kinetic properties of ChEs in eyes, gills, and hepatopancreas, investigating their substrate preferences with different thiocholine esters and their sensitivity to inhibition with selective inhibitors. The results suggest that the studied enzymes are ChEs and not nonspecific esterases, due to their apparent affinity for choline esters, with a distinct preference for the substrate acetylthiocholine, and their high sensitivity to inhibition by eserine sulfate. P. serratus eyes can be considered the best tissue for recovery of ChE enzyme: this tissue is easy to isolate, it expresses high levels of ChE, and the enzyme shows properties of a vertebrate AChE.

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications.

Quantum dots (QDs) are inorganic semiconductor nanocrystals that have unique optoelectronic properties responsible for bringing together multidisciplinary research to impel their potential bioanalytical applications. In recent years, the many remarkable optical properties of QDs have been combined with the ability to make them increasingly biocompatible and specific to the target. With this great development, QDs hold particular promise as the next generation of fluorescent probes. This review describes the developments in functionalizing QDs making use of different bioconjugation and capping approaches. The progress offered by QDs is evidenced by examples on QD-based biosensing, biolabeling, and delivery of therapeutic agents. In the near future, QD technology still faces some challenges towards the envisioned broad bioanalytical purposes.

Semiconductor quantum dots in chemical sensors and biosensors.

Quantum dots are nanometre-scale semiconductor crystals with unique optical properties that are advantageous for the development of novel chemical sensors and biosensors. The surface chemistry of luminescent quantum dots has encouraged the development of multiple probes based on linked recognition molecules such as peptides, nucleic acids or small-molecule ligands. This review overviews the design of sensitive and selective nanoprobes, ranging from the type of target molecules to the optical transduction scheme. Representative examples of quantum dot-based optical sensors from this fast-moving field have been selected and are discussed towards the most promising directions for future research.

Does mercury interact with the inhibitory effect of dichlorvos on Palaemon serratus (Crustacea: Decapoda) cholinesterase?

Mercury is one of the most hazardous metals that may contaminate estuarine ecosystems and induce toxic effects on wildlife organisms. It has been suggested that impairment of cholinesterase (ChE) activity may be involved in the resulting mercury toxicity. Following Palaemon serratus exposure to mercury chloride (HgCl2), no effect on ChE activity was observed whatever the concentration used (to 37.5 microM) or the time of exposure (to 7 days). By contrast, following 24 h exposure to dichlorvos, an organophosphate insecticide with a well-characterised anti-ChE action, decrease of ChE activity was observed until 30 to 40% basal activity, which seems to be the minimum activity required for prawn survival. In addition, HgCl2 does not affect dichlorvos toxicity and treatments with a mixture of both compounds can be interpreted as the sum of the two independent toxicities. Therefore, mercury and insecticide toxicities are independent and ChE activity from P. serratus eyes seems to be a reliable and sensitive biomarker for organophosphate insecticides even when organisms are simultaneously exposed to mercury.

Mechanisms of cholinesterase inhibition by inorganic mercury.

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.

Cholinesterase from the common prawn (Palaemon serratus) eyes: catalytic properties and sensitivity to organophosphate and carbamate compounds.

The main purpose of this study was to describe the kinetic properties of the cholinesterase (ChE) enzyme present in the eyes of the prawn Palaemon serratus, an abundant, ecological and commercially relevant species of European coastal environments. The obtained results suggest that the studied enzyme is a ChE and not a non-specific esterase, due to its apparent affinity for choline esters and the high sensitivity to eserine sulphate. This ChE displays a distinct preference for the substrate acetylthiocholine, showing a triphasic behaviour, with activation at low concentrations and inhibition by excess of substrate. Moreover, irreversible ChE inhibition by several organophosphate and carbamate compounds was characterized. All the irreversible inhibitions were homogeneous following a second-order rate reaction. The bimolecular rate constant (k(i)) values of ChE inhibition by the tested pesticides were also estimated and compared with available data from other invertebrate and vertebrate species. In conclusion, the results of the present study showed that prawn eyes possess only one ChE with typical properties of acetylcholinesterase, which is highly sensitive to the tested anti-cholinesterase compounds.