Antisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucleases and targeting capability via simple surface modification. We constructed an antisense gold-nanobeacon consisting of a stem-looped oligonucleotide double-labelled with 3′-Cy3 and 5′-Thiol-C6 and tested for the effective blocking of gene expression in colorectal cancer cells. Due to the beacon conformation, gene silencing was directly detected as fluorescence increases with hybridisation to target, which can be used to assess the level of silencing. Moreover, this system was extensively evaluated for the genotoxic, cytotoxic and proteomic effects of gold-nanobeacon exposure to cancer cells. The exposure was evaluated by two-dimensional protein electrophoresis followed by mass spectrometry to perform a proteomic profile and 3-(4,5-Dimethylthiazol-2- Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, glutathione-S-transferase assay, micronucleus test and comet assay to assess the genotoxicity. This integrated toxicology evaluation showed that the proposed nanotheranostics strategy does not exhibit significant toxicity, which is extremely relevant when translating into in vivo systems.

Spatial and temporal control of molecular mechanisms can be achieved using photolabile bonds that connect biomolecules to protective caging groups, which can be cleaved upon irradiation of a specific wavelength, releasing the biomolecule ready-to-use. Here we apply and improve a previously reported strategy to tightly control in vitro transcription reactions. The strategy involves two caging molecules that block both ATP and GTP nucleotides. Additionally, we designed a molecular beacon complementary to the synthesized mRNA to infer its presence through a light signal. Upon release of both nucleotides through a specific monochromatic light (390 and 325 nm) we attain a light signal indicative of a successful in vitro transcription reaction. Similarly, in the absence of irradiation, no intense fluorescence signal was obtained. We believe this strategy could further be applied to DNA synthesis or the development of logic gates.

Cancer development is amultistep process in which exosomes play important roles. Exosomes are small vesicles formed in vesicular bodies in the endosomal network. The major role of exosomes seems to be the transport of bioactive molecules between cells. Depending on the cell of origin, exosomes are implicated in the regulation of several cellular events, with phenotypic consequences in recipient cells. Cancer derived exosomes (CCEs) are important players in the formation of the tumour microenvironment by (i) enabling the escape of tumour cells to immunological system and help initiating the inflammatory response; (ii) acting in the differentiation of fibroblasts and mesenchymal cells into myofibroblasts; (iii) triggering the angiogenic process; and (iv) enhancing the metastatic evolution of the tumour by promoting epithelial to mesenchymal transformation of tumour cells and by preparing the tumour niche in the new anatomical location. Since the finding that exosomes content resembles that of the cell of origin, they may be regarded as suitable biomarkers for cancer diagnosis, allowing for diagnosis and prognosis via a minimal invasive procedure. Exosome involvement in cancer may open new avenues regarding therapeutics, such as vectors for targeted drug delivery.

This chapter focuses on several sensing strategies and major recent advances in the use of gold nanoparticles in (bio)sensing of chemical and biological analytes. A brief introduction is presented on relevant properties of gold nanoparticles for sensing, the main types of (bio)chemical sensors, and the main detection techniques, followed by subsections according to sensing methodologies. These include colorimetric sensing and the biobarcode assay, fluorometric-based methods, electric and electrochemical sensing, and, last, more recent and advanced methodologies such as surface plasmon resonance and Raman-based sensors. In closing, relevance is given to advanced methods, featuring extremely high sensitivity and selectivity, down to single-molecule detection. Anisotropic gold nanoparticles have a special role in future developments.

The design of small interfering RNA (siRNA) delivery materials showing efficacy in vivo is at the forefront of nanotherapeutics research. Polyurea (PURE-type) dendrimers are 'smart' biocompatible 3D polymers that unveil a dynamic and elegant back-folding mechanism involving hydrogen bonding between primary amines at the surface and tertiary amines and ureas at the core. Similarly, to a biological proton pump, they are able to automatically and reversibly transform their conformation in response to pH stimulus. Here, we show that PURE-G4 is a useful gene silencing platform showing no cellular toxicity. As a proof of concept we investigated the PURE-G4-siRNA dendriplex, which was shown to be an attractive platform with high transfection efficacy. The simplicity associated with the complexation of siRNA with polyurea dendrimers makes them a powerful tool for efficient cytosolic siRNA delivery.

Nanoenabled technology holds great potential for health issues and biological research. Among the numerous inorganic nanoparticles that are available today, gold nanoparticles are fully developed as therapeutic and diagnostic agents both in vitro and in vivo due to their physicochemical properties. Owing to this, substantial work has been conducted in terms of developing biosensors for noninvasive and targeted tumor diagnosis and treatment. Some studies have even expanded into clinical trials. This article focuses on the fundamentals and synthesis of gold nanoparticles, as well as the latest, most promising applications in cancer research, such as molecular diagnostics, immunosensors, surface-enhanced Raman spectroscopy and bioimaging. Challenges to their further translational development are also discussed.

In the last 30 years we have assisted to a massive advance of nanomaterials in material science. Nanomaterials and structures, in addition to their small size, have properties that differ from those of larger bulk materials, making them ideal for a host of novel applications. The spread of nanotechnology in the last years has been due to the improvement of synthesis and characterization methods on the nanoscale, a field rich in new physical phenomena and synthetic opportunities. In fact, the development of functional nanoparticles has progressed exponentially over the past two decades. This work aims to extensively review 30 years of different strategies of surface modification and functionalization of noble metal (gold) nanoparticles, magnetic nanocrystals and semiconductor nanoparticles, such as quantum dots. The aim of this review is not only to provide in-depth insights into the different biofunctionalization and characterization methods, but also to give an overview of possibilities and limitations of the available nanoparticles.

A novel synthetic methodology for star shaped gold-coated magnetic nanoparticles is reported. The coating is performed in two steps: formation of gold nuclei at the surface of magnetite nanoparticles followed by growth of the gold nuclei into a complete star shaped shell. The star-shaped gold-coated magnetic nanoparticles thus obtained preserve the magnetic properties of the precursor magnetite nanoparticles, e. g. they can be easily separated with a magnet. In addition, the gold coating provides interesting optical properties while simultaneously allowing for biofunctionalization that may be advantageous for biological applications, such as (bio)detection via surface-enhanced Raman spectroscopy (SERS). As a proof-of-concept, a capping agent terminated with a nickel(II)-nitrilotriacetate group showing high affinity for histidine was used to modify the surface of the nanoparticles. The resulting star-shaped nanoparticles were used to selectively capture histidine-tagged maltose-binding protein from a crude cell extract. Finally, the performance of star shaped gold-coated magnetic nanoparticles as SERS platforms was demonstrated through the detection of Raman active dye (Astra Blue).

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.

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.

Traditional methods of drug delivery present several drawbacks, mainly due to off-target effects that may originate severe side and toxic effect to healthy tissues. Parallel to the development of novel more effective drugs, particular effort has been dedicated to develop and optimize drug delivery vehicles capable of specifically targeting the required tissue/organ and to deliver the cargo only where and when it is needed. New drug delivery systems based on nanoscale devices showing new and improved pharmacokinetic and pharmacodynamics properties like enhanced bioavailability, high drug loading or systemic stability have surged in the past decade as promising solutions to the required therapeutic efficacy. Amongst these nanoscale vectors, nanoparticles for drug delivery, such as polymeric, lipidbased, ceramic or metallic nanoparticles, have been at the forefront of pharmaceutical development. The interest in nanomedicine for treatment and diagnosis is clearly reflected on the increasing number of publications and issued patents every year. Here, we provide a broad overview of novel nanoparticle based drug delivery systems, ranging from polymeric systems to metal nanoparticles, while simultaneously listing the most relevant related patents.