FTA-LAMP based biosensor for a rapid in-field detection of Globodera pallida-the pale potato cyst nematode.

The combination of a sensitive and specific magnetoresistive sensing device with an easy DNA extraction method and a rapid isothermal amplification is presented here targeting the on-site detection of Globodera pallida, a potato endoparasitic nematode. FTA-cards were used for DNA extraction, LAMP was the method developed for DNA amplification and a nanoparticle functionalized magnetic-biosensor was used for the detection. The combinatorial effect of these three emerging technologies has the capacity to detect G. pallida with a detection limit of one juvenile, even when mixed with other related species. This combined system is far more interesting than what a single technology can provide. Magnetic biosensors can be combined with any DNA extraction protocol and LAMP forming a new solution to target G. pallida. The probe designed in this study consistently distinguished G. pallida (∆Vac binding/Vac sensor above 1%) from other cyst nematodes (∆Vac binding/Vac sensor below 1%). It was confirmed that DNA either extracted with FTA-cards or Lab extraction Kit was of enough quantity and quality to detect G. pallida whenever present (alone or in mixed samples), ensuring probe specificity and sensitivity. This work provides insights for a new strategy to construct advanced devices for pathogens in-field diagnostics. LAMP runs separately but can be easily integrated into a single device.

Rapid Detection and Identification of Vancomycin-Sensitive Bacteria Using an Electrochemical Apta-Sensor.

In order to combat the complex and diverse infections caused by bacteria, it is essential to develop efficient diagnostic tools. Current techniques for bacterial detection rely on laborious multistep procedures, with high costs and extended time of analysis. To overcome these limitations, we propose here a novel portable electrochemical biosensor for the rapid detection and identification of Gram-positive bacteria that leverages the recognition capabilities of vancomycin and aptamers. A vancomycin-modified screen-printed carbon electrode was used to selectively capture Gram-positive bacteria susceptible to this antibiotic. Electrochemical impedance spectroscopy and scanning electron microscopy demonstrated that capture was achieved in 10 min, with a limit of detection of only 2 CFU/mL. We then tested the device's potential for aptamer-based bacterial identification using Staphylococcus aureus and Bacillus cereus as the test strains. Specifically, electrodes with captured bacteria were exposed to species-specific aptamers, and the resulting changes in current intensity were analyzed using differential pulse voltammetry. When used directly in untreated milk or serum, the system was able to successfully identify a small amount of S. aureus and B. cereus (100 CFU/mL) in less than 45 min. This novel biosensor has the potential to serve as an invaluable tool that could be used, even by inexperienced staff, in a broad range of settings including clinical diagnostics, food safety analysis, environmental monitoring, and security applications.

The Antioxidant and Immunomodulatory Potential of Coccoloba alnifolia Leaf Extracts.

Oxidative stress has been associated with different diseases, and different medicinal plants have been used to treat or prevent this condition. The leaf ethanolic extract (EE) and aqueous extract (AE) from Coccoloba alnifolia have previously been characterized to have antioxidant potential in vitro and in vivo. In this study, we worked with EE and AE and two partition phases, AF (ethyl acetate) and BF (butanol), from AE extract. These extracts and partition phases did not display cytotoxicity. The EE and AE reduced NO production and ROS in all three concentrations tested. Furthermore, it was observed that EE and AE at 500 µg/mL concentration were able to reduce phagocytic activity by 30 and 50%, respectively. A scratch assay using a fibroblast cell line (NHI/3T3) showed that extracts and fractions induced cell migration with 60% wound recovery within 24 h, especially for BF. It was also observed that AF and BF had antioxidant potential in all the assays evaluated. In addition, copper chelation was observed. This activity was previously not detected in AE. The HPLC-DAD analysis showed the presence of phenolic compounds such as p-cumaric acid and vitexin for extracts, while the GNPS annotated the presence of isoorientin, vitexin, kanakugiol, and tryptamine in the BF partition phase. The data presented here demonstrated that the EE, AE, AF, and BF of C. alnifolia have potential immunomodulatory effects, antioxidant effects, as well as in vitro wound healing characteristics, which are important for dynamic inflammation process control.

Exploring the Antioxidant Potential of Talisia esculenta Using In Vitro and In Vivo Approaches.

Medicinal plants, such as Talisia esculenta, are rich in antioxidant biomolecules, which are used in the treatment and prevention of many diseases. The antioxidant potential of T. esculenta extracts obtained from leaves and fruit peels was investigated using biochemical and 3T3 cell line assays as well as in vivo assays using an organism model Tenebrio molitor. Four extracts were tested: hydroethanolic extracts from leaves (HF) and from fruit peels (HC), and infusion extracts from leaves (IF) and from fruit peels (IC). The biochemical assays demonstrated an antioxidant capacity verified by TAC, reducing power, DPPH, and copper chelating assays. None of the extracts exhibited cytotoxicity against 3T3 cells, instead offering a protection against CuSO4-induced oxidative stress. The antioxidant activity observed in the extracts, including their role as free radical scavengers, copper chelators, and stress protectors, was further confirmed by T. molitor assays. The CLAE-DAD analysis detected phenolic compounds, including gallic acid, rutin, and quercitrin, as the main constituents of the samples. This study highlights that leaf and fruit peels extracts of T. esculenta could be effective protectors against ROS and copper-induced stress in cellular and invertebrate models, and they should be considered as coadjutants in the treatment and prevention of diseases related to oxidative stress and for the development of natural nutraceutical products.

Biological and pharmacological aspects of tannins and potential biotechnological applications.

Secondary metabolites are divided into three classes: phenolic, terpenoid, and nitrogenous compounds. Phenolic compounds are also known as polyphenols and include tannins, classified as hydrolysable or condensed. Herein, we explored tannins for their ROS reduction characteristics and role in homeostasis. These activities are associated with the numbers and degree of polymerisation of reactive hydroxyl groups present in the phenolic rings of tannins. These characteristics are associated with anti-inflammatory, anti-aging, and anti-proliferative health benefits. Tannins can reduce the risk of cancer and neurodegenerative diseases, such as cardiovascular diseases and Alzheimer's, respectively. These biomolecules may be used as nutraceuticals to maintain good gut microbiota. Industrial applications include providing durability to leather, anti-corrosive properties to metals, and substrates for 3D printing and in bio-based foam manufacture. This review updates regarding tannin-based research and highlights its biological and pharmacological relevance and potential applications.

A Lab-on-a-Chip Approach for the Detection of the Quarantine Potato Cyst Nematode Globodera pallida.

The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin−streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor's electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor's potential as a bioanalytical device for field applications and border phytosanitary inspections.

Multifunctional Core@Satellite Magnetic Particles for Magnetoresistive Biosensors.

Magnetoresistive (MR) biosensors combine distinctive features such as small size, low cost, good sensitivity, and propensity to be arrayed to perform multiplexed analysis. Magnetic nanoparticles (MNPs) are the ideal target for this platform, especially if modified not only to overcome their intrinsic tendency to aggregate and lack of stability but also to realize an interacting surface suitable for biofunctionalization without strongly losing their magnetic response. Here, we describe an MR biosensor in which commercial MNP clusters were coated with gold nanoparticles (AuNPs) and used to detect human IgG in water using an MR biochip that comprises six sensing regions, each one containing five U-shaped spin valve sensors. The isolated AuNPs (satellites) were stuck onto an aggregate of individual iron oxide crystals (core) so that the resulting core@satellite magnetic particles (CSMPs) could be functionalized by the photochemical immobilization technique-an easy procedure that leads to oriented antibodies immobilized upright onto gold. The morphological, optical, hydrodynamic, magnetic, and surface charge properties of CSMPs were compared with those exhibited by the commercial MNP clusters showing that the proposed coating procedure endows the MNP clusters with stability and ductility without being detrimental to magnetic properties. Eventually, the high-performance MR biosensor allowed us to detect human IgG in water with a detection limit of 13 pM (2 ng mL-1). Given its portability, the biosensor described in this paper lends itself to a point-of-care device; moreover, the features of the MR biochip also make it suitable for multiplexed analysis.

Phenolic Acids as Antidepressant Agents.

Depression is a psychiatric disorder affecting the lives of patients and their families worldwide. It is an important pathophysiology; however, the molecular pathways involved are not well understood. Pharmacological treatment may promote side effects or be ineffective. Consequently, efforts have been made to understand the molecular pathways in depressive patients and prevent their symptoms. In this context, animal models have suggested phytochemicals from medicinal plants, especially phenolic acids, as alternative treatments. These bioactive molecules are known for their antioxidant and antiinflammatory activities. They occur in some fruits, vegetables, and herbal plants. This review focused on phenolic acids and extracts from medicinal plants and their effects on depressive symptoms, as well as the molecular interactions and pathways implicated in these effects. Results from preclinical trials indicate the potential of phenolic acids to reduce depressive-like behaviour by regulating factors associated with oxidative stress, neuroinflammation, autophagy, and deregulation of the hypothalamic-pituitary-adrenal axis, stimulating monoaminergic neurotransmission and neurogenesis, and modulating intestinal microbiota.

Combined detection of molecular and serological signatures of viral infections: The dual assay concept.

The recent worldwide spread of viral infections has highlighted the need for accurate, fast, and inexpensive disease diagnosis and monitorization methods. Current diagnostics tend to focus either on molecular or serological testing. In this work we propose a dual detection assay approach for viral diseases, where both serological and molecular assays are combined in a single analysis performed on a magnetoresistive system. This type of assay guarantees an accurate assessment of the infection phase, saving time and costs associated with multiple independent tests. Zika and dengue viruses were used as model diseases for the validation of the system. Human IgG anti-zika and anti-dengue antibodies were successfully detected in infected patients' serum, using a novel approach combining competitive and sandwich strategies in a magnetoresistive portable platform. Specificity and sensitivity values of 100% were obtained. Calibration curves with dynamic ranges between 10 ng/mL and 1 µg/mL were established achieving LODs of 1.26 and 1.38 nM for IgG anti-ZIKV and anti-DENV antibodies, respectively. Viral RNA detection down to a few hundreds of pM was also successfully carried out after the design of specific oligo probes and primers for RT-PCR amplification. Dual assays were performed for both viruses, where viral RNA and anti-virus antibodies in serum samples were simultaneously detected. The results obtained for the detection of the molecular and serological targets in the dual assay format show no significant difference between the ones obtained individually, proving the feasibility and accuracy of the dual detection assay. This assay format represents a new paradigm in viral infections diagnostics.

Point-of-care quantification of serum cellular fibronectin levels for stratification of ischemic stroke patients.

The abundance of cellular fibronectin (c-Fn) for ischemic stroke patients and the narrow time-window (<4.5 h) for the decision to administer the thrombolytic treatment with recombinant tissue plasminogen activator (rtPA) are challenging for the development of a point-of-care (PoC) diagnostic platform. We report a case of stratification of ischemic stroke patients based on a magnetoresistive biosensor platform that quantifies the c-Fn levels in a small volume of serum, within the clinically relevant time-window. Our PoC platform uses different ratios of biofunctionalized magnetic nanoparticles (MNPs) as immunoassay labels to adjust the sensitivity within the clinically relevant ranges for c-Fn (1-4 µg/mL). After optimizing the detection range, resolution, and sensitivity, our device was able to stratify ischemic stroke patients who developed hemorrhagic transformation, the main side-effect of rtPA, from those (both non-treated and treated with rtPA) who did not.

Multiple Bacteria Identification in the Point-of-Care: an Old Method Serving a New Approach.

The accurate diagnosis of bacterial infections is of critical importance for effective treatment decisions. Due to the multietiologic nature of most infectious diseases, multiplex assays are essential for diagnostics. However, multiplexability in nucleic acid amplification-based methods commonly resorts to multiple primers and/or multiple reaction chambers, which increases analysis cost and complexity. Herein, a polymerase chain reaction (PCR) offer method based on a universal pair of primers and an array of specific oligonucleotide probes was developed through the analysis of the bacterial 16S ribosomal RNA gene. The detection system consisted of DNA hybridization over an array of magnetoresistive sensors in a microfabricated biochip coupled to an electronic reader. Immobilized probes interrogated single-stranded biotinylated amplicons and were obtained using asymmetric PCR. Moreover, they were magnetically labelled with streptavidin-coated superparamagnetic nanoparticles. The benchmarking of the system was demonstrated to detect five major bovine mastitis-causing pathogens: Escherichia coli, Klebsiella sp., Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. All selected probes proved to specifically detect their respective amplicon without significant cross reactivity. A calibration curve was performed for S. agalactiae, which demonstrates demonstrating a limit of detection below 30 fg/µL. Thus, a sensitive and specific multiplex detection assay was established, demonstrating its potential as a bioanalytical device for point-of-care applications.

In Vitro Antioxidant, Anti-Biofilm, and Solar Protection Activities of Melocactus zehntneri (Britton & Rose) Pulp Extract.

Cactaceae plants are important due to their nutritional and therapeutic values. This study aimed to identify the phytochemical profile and biological activities of six Melocactus zehntneri pulp extracts: hexane extract (HE), chloroform extract (CE), ethanol extract (EE), methanol extract (ME), final water extract (FWE), and water extract (WE). Sugar, phenolic compounds, and protein content of the extracts were determined. Then thin layer chromatography (TLC) was performed to detect the presence of terpenes (ursolic and oleanolic acids), saponins, sugars, and glycoproteins. These extracts were analyzed for antioxidant activity via in vitro assay. HE showed 75% ferric chelating activity. All extracts showed 80-100% superoxide and hydroxyl radical-scavenging activities, respectively. Further, all extracts at 25 µg/mL showed 60% activity against DPPH. Moreover, in the 3T3 cells lines, no cytotoxicity was observed; however, therapeutic activity against the effects of the H2O2 treatment was exhibited. Finally, the polar extracts (EE, ME, FWE, and WE), particularly WE, elicited activity against the biofilms of Staphylococcus epidermidis, and HE and CE expressed a capacity for solar protection.

Biosensors for On-Farm Diagnosis of Mastitis.

Bovine mastitis is an inflammation of the mammary gland caused by a multitude of pathogens with devastating consequences for the dairy industry. Global annual losses are estimated to be around €30 bn and are caused by significant milk losses, poor milk quality, culling of chronically infected animals, and occasional deaths. Moreover, mastitis management routinely implies the administration of antibiotics to treat and prevent the disease which poses serious risks regarding the emergence of antibiotic resistance. Conventional diagnostic methods based on somatic cell counts (SCC) and plate-culture techniques are accurate in identifying the disease, the respective infectious agents and antibiotic resistant phenotypes. However, pressure exists to develop less lengthy approaches, capable of providing on-site information concerning the infection, and in this way, guide, and hasten the most adequate treatment. Biosensors are analytical tools that convert the presence of biological compounds into an electric signal. Benefitting from high signal-to-noise ratios and fast response times, when properly tuned, they can detect the presence of specific cells and cell markers with high sensitivity. In combination with microfluidics, they provide the means for development of automated and portable diagnostic devices. Still, while biosensors are growing at a fast pace in human diagnostics, applications for the veterinary market, and specifically, for the diagnosis of mastitis remain limited. This review highlights current approaches for mastitis diagnosis and describes the latest outcomes in biosensors and lab-on-chip devices with the potential to become real alternatives to standard practices. Focus is given to those technologies that, in a near future, will enable for an on-farm diagnosis of mastitis.

Go with the flow: advances and trends in magnetic flow cytometry.

The growing need for biological information at the single cell level has driven the development of improved cytometry technologies. Flow cytometry is a particularly powerful method that has evolved over the past few decades. Flow cytometers have become essential instruments in biomedical research and routine clinical tests for disease diagnosis, prognosis, and treatment monitoring. However, the increasing number of cellular parameters unveiled by genomic, proteomic, and metabolomic data platforms demands an augmented multiplexability. Also, the need for identification and quantification of relevant biomarkers at low levels requires outstanding analytical sensitivity and reliability. In addition, growing awareness of the advantages associated with miniaturization of analytical devices is pushing forward the progress in integrated and compact, microfluidic-based devices at the point-of-care. In this context, novel types of flow cytometers are emerging during the search to tackle these challenges. Notwithstanding the relevance of other promising alternatives to standard optical flow cytometry (e.g., mass cytometry, various optical and electrical microcytometers), this report focuses on a recent microcytometric technology based on magnetic sensors and magnetic particles integrated into microfluidic structures for dynamic bioanalysis of fluid samples-magnetic flow cytometry. Its concept, main developments, targeted applications, as well as the challenges and trends behind this technology are presented and discussed. Graphical abstract ᅟ "Kindly advise whether there is online abstract figure for this paper. If so, kindly resupply.The graphical abstract is correctly supplied.

Combining CXCR4-targeted and nontargeted nanoparticles for effective unassisted in vitro magnetic hyperthermia.

The use of targeted nanoparticles for magnetic hyperthermia (MHT) increases MHT selectivity, but often at the expense of its effectiveness. Consequently, targeted MHT is typically used in combination with other treatment modalities. This work describes an implementation of a highly effective monotherapeutic in vitro MHT treatment based on two populations of magnetic particles. Cells were sequentially incubated with two populations of magnetic particles: nonfunctionalized superparamagnetic nanoparticles and anti-CXCR4-functionalized particles. After removing the excess of free particles, an alternating magnetic field (AMF) was applied to produce MHT. The induced cytotoxicity was assessed at different time-points after AMF application. Complete loss of cell viability was observed 72 h after MHT when the iron loading of the anti-CXCR4-functionalized particles was boosted by that of a nontargeted population. Additionally, induction of necrosis resulted in more efficient cell death than did induction of apoptosis. Achieving a uniquely high effectiveness in monotherapeutic MHT demonstrates the potential of this approach to achieve complete loss of viability of cancer cells while avoiding the side effects of dual-treatment strategies that use MHT only as a sensitizing therapy.

A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female Heterodera glycines cyst formation during infection of Glycine max.

Host-mediated (hm) expression of parasite genes as tandem inverted repeats was investigated as a means to abrogate the formation of mature Heterodera glycines (soybean cyst nematode) female cysts during its infection of Glycine max (soybean). A Gateway-compatible hm plant transformation system was developed specifically for these experiments in G. max. Three steps then were taken to identify H. glycines candidate genes. First, a pool of 150 highly conserved H. glycines homologs of genes having lethal mutant phenotypes or phenocopies from the free living nematode Caenorhabditis elegans were identified. Second, annotation of those 150 genes on the Affymetrix soybean GeneChip allowed for the identification of a subset of 131 genes whose expression could be monitored during the parasitic phase of the H. glycines life cycle. Third, a microarray analyses identified a core set of 32 genes with induced expression (>2.0-fold, log base 2) during the parasitic stages of infection. H. glycines homologs of small ribosomal protein 3a and 4 (Hg-rps-3a [accession number CB379877] and Hg-rps-4 [accession number CB278739]), synaptobrevin (Hg-snb-1 [accession number BF014436]) and a spliceosomal SR protein (Hg-spk-1 [accession number BI451523.1]) were tested for functionality in hm expression studies. Effects on H. glycines development were observed 8 days after infection. Experiments demonstrated that 81-93% fewer females developed on transgenic roots containing the genes engineered as tandem inverted repeats. The effect resembles RNA interference. The methodology has been used here as an alternative approach to engineer resistance to H. glycines.

A portable and autonomous magnetic detection platform for biosensing.

This paper presents a prototype of a platform for biomolecular recognition detection. The system is based on a magnetoresistive biochip that performs biorecognition assays by detecting magnetically tagged targets. All the electronic circuitry for addressing, driving and reading out signals from spin-valve or magnetic tunnel junctions sensors is implemented using off-the-shelf components. Taking advantage of digital signal processing techniques, the acquired signals are processed in real time and transmitted to a digital analyzer that enables the user to control and follow the experiment through a graphical user interface. The developed platform is portable and capable of operating autonomously for nearly eight hours. Experimental results show that the noise level of the described platform is one order of magnitude lower than the one presented by the previously used measurement set-up. Experimental results also show that this device is able to detect magnetic nanoparticles with a diameter of 250 nm at a concentration of about 40 fM. Finally, the biomolecular recognition detection capabilities of the platform are demonstrated by performing a hybridization assay using complementary and non-complementary probes and a magnetically tagged 20mer single stranded DNA target.

A decline in transcript abundance for Heterodera glycines homologs of Caenorhabditis elegans uncoordinated genes accompanies its sedentary parasitic phase.

BACKGROUND: Heterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin) during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia. RESULTS: We developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of C. elegans and H. glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97. CONCLUSION: The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.

Horseradish peroxidase: a valuable tool in biotechnology.

Peroxidases have conquered a prominent position in biotechnology and associated research areas (enzymology, biochemistry, medicine, genetics, physiology, histo- and cytochemistry). They are one of the most extensively studied groups of enzymes and the literature is rich in research papers dating back from the 19th century. Nevertheless, peroxidases continue to be widely studied, with more than 2000 articles already published in 2002 (according to the Institute for Scientific Information). The importance of peroxidases is emphasised by their wide distribution among living organisms and by their multiple physiological roles. They have been divided into three superfamilies according to their source and mode of action: plant peroxidases, animal peroxidases and catalases. Among all peroxidases, horseradish peroxidase (HRP) has received a special attention and will be the focus of this review. A brief description of the three super-families is included in the first section of this review. In the second section, a comprehensive description of the present state of knowledge of the structure and catalytic action of HRP is presented. The physiological role of peroxidases in higher plants is described in the third section. And finally, the fourth section addresses the applications of peroxidases, especially HRP, in the environmental and health care sectors, and in the pharmaceutical, chemical and biotechnological industries.