Field-effect-based devices are becoming a basic structural element in a new generation of microbiosensors. Reliable molecular characterization of DNA and/or RNA is of paramount importance for disease diagnostics and to follow up alterations in gene expression profiles. The use of such devices for point-of-need diagnostics has been hindered by the need of standard or real-time PCR amplification procedures. The present work focuses on the development of a tantalum pentoxide (Ta2O5) based sensor for the real-time label free detection of DNA amplification via loop mediated isothermal amplification (LAMP) allowing for quantitative analysis of the cMYC proto-oncogene. The strategy based on the field effect sensor was tested within a range of 1×108–1011 copies of target DNA, and a linear relationship between the log copy number of the initial template DNA and threshold time was observed allowing for a semi-quantitative analysis of DNA template. The concept offers many of the advantages of isothermal quantitative real-time DNA amplification in a label free approach and may pave the way to point-of-care quantitative molecular analysis focused on ease of use and low cost.

pH is a vital physiological parameter that can be used for disease diagnosis and treatment as well as in monitoring other biological processes. Metal/metal oxide based pH sensors have several advantages regarding their reliability, miniaturization, and cost-effectiveness, which are critical characteristics for in vivo applications. In this work, WO3 nanoparticles were electrodeposited on flexible substrates over metal electrodes with a sensing area of 1 mm2. These sensors show a sensitivity of ?56.7 ± 1.3 mV/pH, in a wide pH range of 9 to 5. A proof of concept is also demonstrated using a flexible reference electrode in solid electrolyte with a curved surface. A good balance between the performance parameters (sensitivity), the production costs, and simplicity of the sensors was accomplished, as required for wearable biomedical devices.

The effect of post-deposition annealing temperature on the pH sensitivity of room temperature RF sputtered Ta2O5 was investigated. Structural and morphological features of these films were analyzed before and after annealing at various temperatures. The deposited films are amorphous up to 600 degrees C and crystallize at 700 degrees C in an orthorhombic phase. Electrolyte-insulator-semiconductor (EIS) field effect based sensors with an amorphous Ta2O5 sensing layer showed pH sensitivity above 50 mV/pH. For sensors annealed above 200 degrees C pH sensitivity decreased with increasing temperature. Stabilized sensor response and maximum pH sensitivity was achieved after low temperature annealing at 200 degrees C, which is compatible with the use of polymeric substrates and application as sensitive layer in oxides TFT-based sensors.

We have projected and fabricated a microfluidic platform for DNA sensing that makes use of an optical colorimetric detection method based on gold nanoparticles. The platform was fabricated using replica moulding technology in PDMS patterned by high-aspect-ratio SU-8 moulds. Biochips of various geometries were tested and evaluated in order to find out the most efficient architecture, and the rational for design, microfabrication and detection performance is presented. The best biochip configuration has been successfully applied to the DNA detection of Mycobacterium tuberculosis using only 3 mu l on DNA solution (i.e. 90 ng of target DNA), therefore a 20-fold reduction of reagents volume is obtained when compared with the actual state of the art.

We previously reported a strategy that combines Forster resonance energy transfer (FRET) based spectral codification with a single base extension (SBE) reaction for single nucleotide sequence discrimination in solution. This strategy is capable of unequivocally detect the allele variants present in solution. To extend the use of this tool to any locus of interest, it would be required the development of an universal approach capable of combining a sequence specific SBE primer to an universal sequence labeled and optimized for spectral codification.Here, we extend this concept to a general strategy by means of a labeled universal oligonucleotide primer (donor), a sequence specific primer that allows for incorporation of the complementary acceptor labeled ddNTP, which allows discrimination the allele variant in the sample via the unambiguous FRET signature of the donor/acceptor pair

Up to now, functionalized gold nanoparticles have been optimized as an effective intracellular invitro delivery vehicle for siRNAs to interfere with the expression of specific genes by selective targeting, and provide protection against nucleases. Few examples however of suchlike invivo applications have been described so far. In this study, we report the use of siRNA/RGD gold nanoparticles capable of targeting tumor cells in a lung cancer syngeneic orthotopic murine model. Therapeutic RGD-nanoparticle treatment resulted in successful targeting evident from significant c-myc oncogene down-regulation followed by tumor growth inhibition and prolonged survival of lung tumor bearing mice, possibly via αvβ3 integrin interaction. Our results suggest that RGD gold nanoparticles-mediated delivery of siRNA by intratracheal instillation in mice leads to successful suppression of tumor cell proliferation and respective tumor size reduction. These results reiterate the capability of functionalized gold nanoparticles for targeted delivery of siRNA to cancer cells towards effective silencing of the specific target oncogene. What is more, we demonstrate that the gold-nanoconjugates trigger a complex inflammatory and immune response that might promote the therapeutic effect of the RNAi to reduce tumor size with low doses of siRNA.

sistema de dete{\c c}ão e quantifica{\c c}ão de matéria biológica constituído por um ou mais sensores óticos e uma ou mais fontes luminosas, processo associado e aplica{\c c}ões relacionadas a inven{\c c}ão atual relaciona- se a um sistema e a um processo para a dete{\c c}ão e/ou a identifica{\c c}ão qualitativa e quantitativa do material biológico, tal como seqüências específicas de ácidos nucleicos ou de proteínas como anticorpos, presente em amostras biológicas. o sistema é composto por uma ou mais fontes luminosas ( 1) combinadas com um ou mais fotosensores óticos integrados, ou não, e vários componentes eletrônicos ( 4) , necessários para obter/processar o sinal emitido pelas nanosondas de metal funcionalizadas com uma solu{\c c}ão de compósi to biológico, assim como igualmente um microcontrolador e um microprocessador, reparados ou portátil. esta estrutura do fotosensor pode detectar e determinar as varia{\c c}ões da cor produzidas por nanosondas do metal, sendo este preferencialmente ouro, funcionalizado pelos oligonucleotídeos complementares às seqüências específicas, as proteínas de dna/rna, como por exemplo os anticorpos e/ou os antígenos relativos a determinada doen{\c c}a, ou a outra amostra ou solu{\c c}ão de composto biológico, que devem ser investigada. a dete{\c c}ão e o processo da quantifica{\c c}ão são baseados na resposta de um fotosensor, singular ou integrados, baseado na tecnologia da película fina de silicones amorfos, nanocristalinos ou microcristalino e suas ligas, assim como os semicondutores cerâmicos ativos novos, amorfos e não amorfos.

This protocol describes the synthesis and detailed calibration of a gold nanoparticle-based nanobeacon (Au-nanobeacon) as an innovative theranostic approach for detection and inhibition of sequence-specific DNA and RNA for in vitro and ex vivo applications. Under hairpin configuration, proximity to gold nanoparticles leads to fluorescence quenching; hybridization to a complementary target restores fluorescence emission due to the gold nanobeacons’ conformational reorganization that causes the fluorophore and the AuNP to part from each other. This concept can easily be extended and adapted to assist the in vitro evaluation of silencing potential of a given sequence to be later used for ex vivo gene silencing and RNAi approaches, with the ability to monitor real-time gene delivery action. The time range for the entire protocol is  8 days, including synthesis, functionalization and calibration of Au-nanobeacons, RNAi and gene silencing assays.

Background: Tuberculosis accounted for 8.7 million new cases in 2011 and continues to be one of the leading human infectious diseases. Burdensome is the increasing rate of multi-drug resistant tuberculosis (MDRTB) and the difficulties created for treatment and public health control programs, especially in developing countries. Resistance to rifampicin (RIF), a first line antibiotic, is commonly associated with point mutations within the rpoB gene of Mycobacterium tuberculosis (Mtb) whose detection is considered the best early molecular predictor for MDRTB. Gold nanoparticles functionalized with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for Mtb and single base alterations associated with antibiotic resistance, namely in rpoB gene associated to RIF resistance.Results: We developed a strategy based on the isothermal amplification of sample DNA (LAMP) coupled to specific Au-nanoprobes capable of identifying members of the Mtb complex (MTBC) and discriminating specific mutations within the rpoB gene. Integration of LAMP and Au-nanoprobe assay allowed to detect MTBC member and identify mutations linked to RIF resistance. A total of 12 biological samples were tested and a 100% specificity and sensitivity was attained.Conclusions: There is an increasing demand for simple, fast and cheap methods for the molecular identification of Mtb and for the detection of molecular tags associated to drug resistance suitable for use at point-of-need. Here we describe such a method, that as the potential to get molecular diagnostic of tuberculosis to remote environments.

The potential of a single molecular nanoconjugate to intersect all RNA pathways: from gene specific downregulation to silencing the silencers, i.e. siRNA and miRNA pathways, is demonstrated. Gold-nanobeacons are capable of efficiently silencing single gene expression, exogenous siRNA and endogenous miRNAs while yielding a quantifiable fluorescence signal directly proportional to the level of silencing. The silencing potential is comparable to that of traditional siRNA but the same nanoconjugates structure is also capable of reversing the effect of an exogenous siRNA. We further demonstrate the Gold-nanobeacons' efficiency at targeting and silencing miR-21, an endogenous miRNA involved in cancer development, which could become a valid nanotheranostics approach. Again, expression of miR-21 was inhibited with concomitant increase of the Au-nanobeacons' fluorescence that can be used to assess the silencing effect. This way, a single nanostructure can be used to intersect all RNA regulatory pathways while allowing for direct assessment of effective silencing and cell localization via a quantifiable fluorescence signal, making cancer nanotheranostics possible.

The paper aims to understand the degree of transition towards e-mobility. The assumption is that the degree of convergence between actors of each system (batteries, vehicles, grid, policies, business models and consumers) is an indicator of changes in the present socio-technical regime. After an introduction to the socio-technical transition towards e-mobility, the paper presents and discusses three technology assessment approaches to several projects related to technology, society and politics. There are several thematic crossovers between all projects presented leading to a synergetic technology assessment. This output results from the overlapping areas between the cases and can be used to first assess the extent of changes in the present socio-technical regime, as well as to extract standards and regulations, acceptance/risk analyses and behaviour changes that could be significant in the context of a transition towards electric mobility.

The paper aims to understand the degree of transition towards e-mobility. The assumption is that the degree of convergence between actors of each system (batteries, vehicles, grid, policies, business models and consumers) is an indicator of changes in the present socio-technical regime. After an introduction to the socio-technical transition towards e-mobility, the paper presents and discusses three technology assessment approaches to several projects related to technology, society and politics. There are several thematic crossovers between all projects presented leading to a synergetic technology assessment. This output results from the overlapping areas between the cases and can be used to first assess the extent of changes in the present socio-technical regime, as well as to extract standards and regulations, acceptance/risk analyses and behaviour changes that could be significant in the context of a transition towards electric mobility.

Purpose: To assess P-glycoprotein (P-gp)-modulation ability and the mechanisms of P-gp inhibition mediated by a new synthetic rifampicin derivative, 1,8-dibenzoyl-rifampicin (DiBenzRif), in an in vitro model of the blood-brain barrier (BBB), RBE4 cells, and in membrane mimetic models (liposomes). Methods: P-gp expression (western blot) and activity {[}rhodamine 123 accumulation studies] were assessed until 72 h of exposure to DiBenzRif. The effects on intracellular ATP levels and on P-gp ATPase activity were studied using luciferin-luciferase bioluminescence assay. Membrane fluidity changes were tracked by steady-state anisotropy measurements. Non-P-gp-related rhodamine 123 accumulation was evaluated using liposomes prepared with the main lipids present in RBE4 cell membranes. Results: A significant increase in intracellular rhodamine 123 content was observed in DiBenzRif-treated cells at all tested time-points. This effect was associated with a significant reduction in ATP intracellular levels, the inhibition of P-gp ATPase activity and a significant increase in membrane fluidity. DiBenzRif also favoured rhodamine 123 accumulation in a liposomal model of RBE4 cells, suggesting that it may be useful in increasing intracellular levels of substances that passively diffuse into the cells. Conclusion: DiBenzRif-induced inhibitory effect on P-gp increases xenobiotic accumulation in BBB cells, which may contribute to the development of therapeutic adjuvants to enhance brain penetration of drugs. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Nanotechnology has emerged as a {"}disruptive technology{"} that may provide researchers with new and innovativeways to diagnose, treat and monitor cancer. In fact, nanomedicine approaches have delivered several strategies, suchas new imaging agents, real-time assessments of therapeutic and surgical efficacy, multifunctional, targeted devices capableof bypassing biological barriers to target and silence specific pathways in tumours. Of particular interest, has been theincreased capability to deliver multiple therapeutic agents directly to bulk cancer cells and cancer stem cells that play acritical role in cancer growth and metastasis. These multifunctional targeted nanoconjugates are also capable of avoidingcancer resistance and monitor predictive molecular changes that open the path for preventive action against pre-cancerouscells, minimizing costs and incidence of relapses. A myriad of nanoconjugates with effective silencing and site-targetingmoieties can be developed by incorporating a diverse selection of targeting, diagnostic, and therapeutic components. Adiscussion of the integrative effort of nanotechnology systems with recent developments of biomolecular interactions incancer progression is clearly required. Here, we will update the state of the art related to the development and applicationsof nanoscale platforms and novel biomolecular players in cancer diagnosis, imaging and treatment.

Under laser radiation, cells labeled with gold nanoparticles (AuNPs) are believed to suffer thermal damage due to the transfer of the absorbed light from theAuNPsto the cells. This process, which involves complex mechanisms such as the rapid electron–phonon decay in theAuNPs, followed by phonon–phonon relaxation, culminates in the localized heating of both theAuNPsand the cells, setting the rational for the use of these nanostructures, under laser light, in cancer photothermal therapy (PTT). Here, we discuss the chemical and biological aspects of this promising new therapeutic approach, including the advantages over conventional cancer therapies and the challenges that scientists still need to overcome to progress toward translation research.Read More:http://www.worldscientific.com/doi/abs/10.1142/S179398441330001X

We report a method centred on gold nanoparticle-based surface-assisted laser desorption/ionisation for analysis of deoxynucleotides and alkylated nucleobases. Gold nanoparticles allow for enhanced analysis capability by eliminating undesired signature peaks; thus more elegant mass spectra can be attained that allow identification by nucleotide mass fingerprint. The resulting fingerprinting patterns on the spectra are compared and associated with the presence of different nucleotides in the sample. This method can be easily extended to modified nucleotides implicated in genome lesions due to exposure to environment chemicals, such as DNA adducts (e.g. guanine adducts). The use of gold nanoparticles for surface-assisted laser desorption/ionisation can be an useful tool to resolve common issues of background noise when analysing nucleic acids samples.

P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat's blood-brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre-or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp.