Rapid SARS-CoV-2 sensing through oxygen reduction reaction catalysed by Au@Pt/Au core@shell nanoparticles.

The development of rapid, accurate, sensitive, and low-cost diagnostic methods for COVID-19 detection in real-time is the unique way to control infection sources and monitor illness progression. In this work, we propose an electrochemical biosensor for the rapid and accuracy diagnosis of COVID-19, through the determination of ORF1ab specific sequence. The biosensor is based on the immobilization of a thiolated sequence partially complementary (domain 1) to ORF1ab on gold screen-printed electrodes and the use of bifunctional Au@Pt/Au core@shell nanoparticles modified with a second thiolated sequence partially complementary to ORF1ab (domain 2) as electrochemical indicator of the hybridization of DNA sequences. The synthesized Au@Pt/Au nanoparticles consist of an Au core, a shell of Pt (Au@Pt NPs), that provides an excellent electrocatalytic activity toward the oxygen reduction reaction (ORR) even after formation of hybrid biomaterials by modification, through the Au protuberances growth on the NPs surface, with an oligonucleotide with recognition ability. The ORR electrochemical activity, enhanced by the label element (Au@Pt/Au NPs), has been employed, for the first time, as indicator of the hybridization event. Based on this strategy, target sequences of the SARS-CoV-2 virus have been detected with a detection limit of 32 pM. The selectivity of the biosensor was confirmed by analysing ORF1ab sequence in the presence of DNA sequences from other viruses. The biosensor has been successfully applied to the direct detection of the virus in non-amplified samples of nasopharyngeal swabs from infected and non-infected patients. Results compare well with those obtained through RT-qPCR but our method is more rapid since does not need any amplification process.

Next-generation sequencing reveals that miR-16-5p, miR-19a-3p, miR-451a, and miR-25-3p cargo in plasma extracellular vesicles differentiates sedentary young males from athletes.

A sedentary lifestyle and Olympic participation are contrary risk factors for global mortality and incidence of cancer and cardiovascular disease. Extracellular vesicle miRNAs have been described to respond to exercise. No molecular characterization of young male sedentary people versus athletes is available; so, our aim was to identify the extracellular vesicle miRNA profile of chronically trained young endurance and resistance male athletes compared to their sedentary counterparts. A descriptive case-control design was used with 16 sedentary young men, 16 Olympic male endurance athletes, and 16 Olympic male resistance athletes. Next-generation sequencing and RT-qPCR and external and internal validation were performed in order to analyze extracellular vesicle miRNA profiles. Endurance and resistance athletes had significant lower levels of miR-16-5p, miR-19a-3p, and miR-451a compared to sedentary people. Taking all together, exercise-trained miRNA profile in extracellular vesicles provides a differential signature of athletes irrespective of the type of exercise compared to sedentary people. Besides, miR-25-3p levels were specifically lower in endurance athletes which defines its role as a specific responder in this type of athletes. In silico analysis of this profile suggests a role in adaptive energy metabolism in this context that needs to be experimentally validated. Therefore, this study provides for the first time basal levels of circulating miRNA in extracellular vesicles emerge as relevant players in intertissue communication in response to chronic exercise exposure in young elite male athletes.

Electrochemical monitoring of enzymatic cleavage in nanochannels with nanoparticle-based enhancement: determination of MMP-9 biomarker.

For the first time the use of nanoparticles as carriers of an enzymatic substrate immobilized inside nanoporous alumina membranes is proposed with the aim of amplifying the nanochannel blocking produced and, consequently, improving the efficiency of an enzyme determination through enzymatic cleavage. Streptavidin-modified polystyrene nanoparticles (PSNPs) are proposed as carrier agents, contributing to the steric and the electrostatic blockage due to the charge they present at different pH values. Electrostatic blockage is the predominant effect that governs the blockage in the interior of the nanochannel and is dependent not just in the charge inside the channel, but also in the polarity of the redox indicator used. Hence, the effect of using negatively charged ([Fe(CN)6]4-) and positively charged ([Ru(NH3)6]3+) redox indicator ions is studied for the first time. Under the optimum conditions, matrix-metalloproteinase 9 (MMP-9) is detected at clinically relevant levels (100-1200 ng/mL) showing a detection limit of 75 ng/mL and a quantification limit of 251 ng/mL with good reproducibility (RSD: 8%) and selectivity, also showing an excellent performance in real samples with acceptable recovery percentages (in the range around 80-110%). Overall, our approach represents a cheap and fast sensing methodology of great potential in point-of-care diagnostics.

Bifunctional Au@Pt/Au nanoparticles as electrochemiluminescence signaling probes for SARS-CoV-2 detection.

A novel immunosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) for the sensitive determination of N protein of the SARS-CoV-2 coronavirus is described. For this purpose, bifunctional core@shell nanoparticles composed of a Pt-coated Au core and finally decorated with small Au inlays (Au@Pt/Au NPs) have been synthesized to act as ECL acceptor, using [Ru (bpy)3]2+ as ECL donor. These nanoparticles are efficient signaling probes in the immunosensor developed. The proposed ECL-RET immunosensor has a wide linear response to the concentration of N protein of the SARS-CoV-2 coronavirus with a detection limit of 1.27 pg/mL. Moreover, it has a high stability and shows no response to other proteins related to different virus. The immunosensor has achieved the quantification of N protein of the SARS-CoV-2 coronavirus in saliva samples. Results are consistent with those provided by a commercial colorimetric ELISA kit. Therefore, the developed immunosensor provides a feasible and reliable tool for early and effective detection of the virus to protect the population.

Advances in the screening of antimicrobial compounds using electrochemical biosensors: is there room for nanomaterials?

The abusive use of antimicrobial compounds and the associated appearance of antimicrobial resistant strains are a major threat to human health. An improved antimicrobial administration involves a faster diagnosis and detection of resistances. Antimicrobial susceptibility testing (AST) are the reference techniques for this purpose, relying mainly in the use of culture techniques. The long time required for analysis and the lack of reproducibility of these techniques have fostered the development of high-throughput AST methods, including electrochemical biosensors. In this review, recent electrochemical methods used in AST have been revised, with particular attention on those used for the evaluation of new drug candidates. The role of nanomaterials in these biosensing platforms has also been questioned, inferring that it is of minor importance compared to other applications.

Electrical monitoring of infection biomarkers in chronic wounds using nanochannels.

Chronic wounds represent an important healthcare challenge in developed countries, being wound infection a serious complication with significant impact on patients' life conditions. However, there is a lack of methods allowing an early diagnosis of infection and a right decision making for a correct treatment. In this context, we propose a novel methodology for the electrical monitoring of infection biomarkers in chronic wound exudates, using nanoporous alumina membranes. Lysozyme, an enzyme produced by the human immune system indicating wound infection, is selected as a model compound to prove the concept. Peptidoglycan, a component of the bacterial layer and the native substrate of lysozyme, is immobilized on the inner walls of the nanochannels, blocking them both sterically and electrostatically. The steric blocking is dependent on the pore size (20-100 nm) and the peptidoglycan concentration, whereas the electrostatic blocking depends on the pH. The proposed analytical method is based on the electrical monitoring of the steric/electrostatic nanochannels unblocking upon the specific degradation of peptidoglycan by lysozyme, allowing to detect the infection biomarker at 280 ng/mL levels, which are below those expected in wounds. The low protein adsorption rate and thus outstanding filtering properties of the nanoporous alumina membranes allowed us to discriminate wound exudates from patients with both sterile and infected ulcers without any sample pre-treatment usually indispensable in most diagnostic devices for analysis of physiological fluids. Although size and charge effects in nanochannels have been previously approached for biosensing purposes, as far as we know, the use of nanoporous membranes for monitoring enzymatic cleavage processes, leading to analytical systems for the specific detection of the enzymes has not been deeply explored so far. Compared with previously reported methods, our methodology presents the advantages of no need of neither bioreceptors (antibodies or aptamers) nor competitive assays, low matrix effects and quantitative and rapid analysis at the point-of-care, being also of potential application for the determination of other protease biomarkers.

Enhancing the electrocatalytic activity of palladium nanocluster tags by selective introduction of gold atoms: Application for a wound infection biomarker detection.

In this work, an unprecedented study exploring the role that slight changes into the Pd/Au proportion have in the electrocatalytic activity of bimetallic Pd-AuNPs toward the oxygen reduction reaction (ORR) is conducted. In particular, a careful control of the amount of Au atoms introduced in the cluster and the evaluation of the optimum Pd:Au ratio for getting the maximum catalytic activity is performed for the first time. First, PdNPs are synthesized by alcohol reduction in the presence of polyvinylpyrrolidone, and gold atoms are selectively introduced on vertex or corner positions of the cluster in different amounts following a galvanic substitution procedure. Average elemental analysis done relying on EDX spectroscopy allows to evaluate the Pd:Au ratio in the Pd-AuNPs obtained. Lineal sweep voltammetry and chronoamperometry are used for the evaluation of the Pd-AuNPs electrocatalytic activity toward ORR at a neutral pH compared to PdNPs and AuNPs alone. Our results indicate that, the synergy between both metals is strongly enhanced when the amount of gold is controlled and occupies the more reactive positions of the cluster, reaching a maximum activity for the NPs containing a 30% of gold, while an excess of this metal leads to a decrease in such activity, as a shelter of the PdNPs is achieved. Chronoamperometric analysis allows the quantification of the optimal Pd-AuNPs at over 6 × 109 NPs/mL levels. Such optimal Pd-AuNPs were used as tags, taking advantage of the bio-functionalities of gold present in the cluster, in a proof-of-concept electrochemical immunosensor for the detection of hyaluronidase wound infection biomarker, using magnetic beads as platforms. Hyaluronidase was detected at levels as low as 50 ng/mL (0.02 U/mL; 437 U/mg) with good reproducibility (RSD below 8%) and selectivity (evaluated against bovine serum albumin, immunoglobulin G and lysozyme). The low matrix effects inherent to the use of magnetic bead platforms allowed us to discriminate between wound exudates with both sterile and infected ulcers without sample pre-treatment. This novel electrocatalytic immunoassay has the advantage, over common methods for NP tags electrochemical detection, of the signal generation in the same neutral medium where the immunoassay takes place (10 mM PBS pH 7.4), avoiding the use of additional and hazardous reagents, bringing it closer to their use as point-of-care devices. Overall, our findings may be of great interest not only for biosensing, but also for applications such as energy converting on fuel cells, in which the ORR has a pivotal role.

Unfolded p53 as a Marker of Oxidative Stress in Mild Cognitive Impairment, Alzheimer's and Parkinson's Disease.

AIMS: There are several candidate biomarkers for AD and PD which differ in sensitivity, specificity, cost-effectiveness, invasiveness, logistical and technical demands. This study is aimed to test whether plasma concentration of unfolded p53 may help to discriminate among the neurodegenerative processes occurring in Mild Cognitive Impairment, Alzheimer's disease and Parkinson's disease. METHODS: An electrochemical immunosensor was used to measure unfolded p53 in plasma samples of 20 Mild Cognitive Impairment (13 males/7 females; mean age 74.95±5.31), 20 Alzheimer's (11 males/9 females; mean age: 77.25±7.79), 15 Parkinson's disease patients (12 males/3 females; mean age: 68.60 ± 7.36) and its respective age/sex/studies-matched controls. RESULTS: We observed a significantly higher concentration of unfolded p53 in the plasma of patients of each of the three pathologies with respect to their control groups (p=0.000). Furthermore, the plasma concentration of unfolded p53 was significantly higher in Alzheimer's disease patients in comparison with Mild Cognitive Impairment patients (p=0.000) and Parkinson's disease patients (p=0.006). No significant difference between Mild Cognitive Impairment and Parkinson's disease patients was observed (p=0.524). CONCLUSION: Our results suggest that unfolded p53 concentration in the plasma may be a useful biomarker for an undergoing neuropathological process that may be common, albeit with different intensity, to different diseases.

Electrochemical Biosensors Based on Nanomaterials for Early Detection of Alzheimer's Disease.

Alzheimer's disease (AD) is an untreatable neurodegenerative disease that initially manifests as difficulty to remember recent events and gradually progresses to cognitive impairment. The incidence of AD is growing yearly as life expectancy increases, thus early detection is essential to ensure a better quality of life for diagnosed patients. To reach that purpose, electrochemical biosensing has emerged as a cost-effective alternative to traditional diagnostic techniques, due to its high sensitivity and selectivity. Of special relevance is the incorporation of nanomaterials in biosensors, as they contribute to enhance electron transfer while promoting the immobilization of biological recognition elements. Moreover, nanomaterials have also been employed as labels, due to their unique electroactive and electrocatalytic properties. The aim of this review is to add value in the advances achieved in the detection of AD biomarkers, the strategies followed for the incorporation of nanomaterials and its effect in biosensors performance.