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.

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.

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.

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.

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.

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.

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

Tuberculosis (TB) remains one of the most serious infectious diseases in the world and the rate of new cases continues to increase. The development of cheap and simple methodologies capable of identifying TB causing agents belonging to the Mycobacterium tuberculosis Complex (MTBC), at point-of-need, in particular in resource-poor countries where the main TB epidemics are observed, is of paramount relevance for the timely and effective diagnosis and management of patients. TB molecular diagnostics, aimed at reducing the time of laboratory diagnostics from weeks to days, still require specialised technical personnel and labour intensive methods. Recent nanotechnology-based systems have been proposed to circumvent these limitations. Here, we report on a paper-based platform capable of integrating a previously developed Au-nanoprobe based MTBC detection assay-we call it {"}Gold on Paper{"}. The Au-nanoprobe assay is processed and developed on a wax-printed microplate paper platform, allowing unequivocal identification of MTBC members and can be performed without specialised laboratory equipment. Upon integration of this Au-nanoprobe colorimetric assay onto the 384-microplate, differential colour scrutiny may be captured and analysed with a generic {"}smartphone{"} device. This strategy uses the mobile device to digitalise the intensity of the colour associated with each colorimetric assay, perform a Red Green Blue (RGB) analysis and transfer relevant information to an off-site lab, thus allowing for efficient diagnostics. Integration of the GPS location metadata of every test image may add a new dimension of information, allowing for real-time epidemiologic data on MTBC identification.

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).

Sistema para detección, identificación y cuantificación en material biológico, compuesto por una o más fuentes de luz (1) combinado con uno o más fotosensores ópticos (6 y 7) y diversos componentes electrónicos (4), necesarios para obtener/procesar la señal emitida caracterizado por: a) La fuente de luz (1), pulsada (2) o no, compuesta de láseres de estado sólido de baja energía o diodos emisores de luz, cuyo rango de longitud de onda está localizado entre 400 y 800 nm con una intensidad de luminosidad controlable que varía entre los valores de 0.01 mW/cm 2 y 100 mW/cm 2 ; b) El fotosensor, sencillo (6 y 7a) y (6 y 7b) o integrado (6, 4 y 7) compuesto de películas delgadas de silicio amorfo o nanocristalino o microcristalino y/o por semiconductores de cerámica tales como IGZO, IAgZO, SnZIO, GZIO, CuOIZ, GITO, entre otros, y basado en estructuras tipo pi'ii'n o MIS, que funciona en un rango de longitudes de onda desde el infrarrojo hasta el ultravioleta, y prové una información cualitativa y cuantitativa basada en la hibridización especifica y selectiva de sondas funcionalizadas con nanopartículas de metal; c) Siendo provista la eliminación del sistema a través de una fuente de energía convencional o a través de baterías fotovoltaicas, que dan portabilidad al sistema, siendo focalizada la luz emitida sobre la muestra, preferiblemente utilizando microlentes, siendo la muestra o muestras no fijadas físicamente al sensor o sensores, colocando la muestra biológica referida (5) sobre el lado opuesto (6) del sustrato donde se deposita el fotosensor (6 y 7).

We present a new approach for real-time monitoring of PCR amplification of a specific sequence from the human c-MYC proto-oncogene using a Ta(2)O(5) electrolyte-insulator-semiconductor (EIS) sensor. The response of the fabricated EIS sensor to cycle DNA amplification was evaluated and compared to standard SYBR-green fluorescence incorporation, showing it was possible to detect DNA concentration variations with 30 mV/μM sensitivity. The sensor's response was then optimized to follow in real-time the PCR amplification of c-MYC sequence from a genomic DNA sample attaining an amplification profile comparable to that of a standard real-time PCR. Owing to the small size, ease of fabrication and low-cost, the developed Ta(2)O(5) sensor may be incorporated onto a microfluidic device and then used for real-time PCR. Our approach may circumvent the practical and economical obstacles posed by current platforms that require an external fluorescence detector difficult to miniaturize and incorporate into a lab-on-chip system.

In this paper we report on the fabrication and performance of a portable and low cost optoelectronic platform integrating a double color tuned light emitting diode as light source, an amorphous/nanocrystalline silicon photodetector with a flat spectral response in the wavelength range from 520. nm to 630. nm and integrated electronic for signal acquisition and conditioning constituted by current to voltage converter, a filter and an amplification stage, followed by an analog to digital converter, with appropriate software for full automation to minimize human error. Incorporation of the double color tuned light emitting diode provides for a simple yet innovative solution to signal acquisition independently from the light intensity and/or solution concentration, while considerably decreasing production costs. Detection based on Au-nanoprobes constitutes the biorecognition step and allowed identification of specific sequences of Mycobacterium tuberculosis complex, namely Mycobacterium bovis and M. tuberculosis in biological samples.

The aim of this chapter is to provide an overview of the available and emerging molecular diagnostic methods that take advantage of the unique nanoscale properties of nanoparticles (NPs) to increase the sensitivity, detection capabilities, ease of operation, and portability of the biodetection assemblies. The focus will be on noble metal NPs, especially gold NPs, fluorescent NPs, especially quantum dots, and magnetic NPs, the three main players in the development of probes for biological sensing. The chapter is divided into four sections: a first section covering the unique physicochemical properties of NPs of relevance for their utilization in molecular diagnostics; the second section dedicated to applications of NPs in molecular diagnostics by nucleic acid detection; and the third section with major applications of NPs in the area of immunoassays. Finally, a concluding section highlights the most promising advances in the area and presents future perspectives.

The present invention relates to a colorimetric method for the detection of specific nucleic acids sequences, including mutations or single nucleotide polymorphisms within nucleic acid sequences, through the aggregation of nanoparticles functionalized with modified oligonucleotides, induced by an increase of the medium's ionic strength. Another aspect of the present invention relates with the development of a kit based on the method of the present invention, allowing for a quick and easy detection of specific nucleic acids sequences, including mutations or single nucleotide polymorphisms within nucleic acid sequences.

The derivatization of gold-silver alloy nanoparticles with thiol-ssDNA oligonucleotides (AuAg-alloy-nanoprobes) and their use in nucleic acid detection is presented. A non-cross-linking method has been previously developed by our group using gold nanoparticles, which is based on the colorimetric comparison of solutions before and after salt-induced nanoprobe aggregation. Only the presence of a complementary target stabilizes the nanoprobe, preventing aggregation and colorimetric change after salt addition. Through this approach, the AuAg-alloy-nanoprobes allowed to specifically detect a sequence derived from the RNA polymerase β-subunit gene of Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, with a 2.5-fold enhanced sensitivity (0.3 μg of total DNA) when compared to their gold counterparts.

The present invention relates to a colorimetric method for the detection of specific nucleic acids sequences, including mutations or single nucleotide polymorphisms within nucleic acid sequences, through the aggregation of nanoparticles functionalized with modified oligonucleotides, induced by an increase of the medium's ionic strength. Another aspect of the present invention relates with the development of a kit based on the method of the present invention, allowing for a quick and easy detection of specific nucleic acids sequences, including mutations or single nucleotide polymorphisms within nucleic acid sequences.

A dye sensitized TiO2 photodetector has been integrated with a DNA detection method based on non-cross-linking hybridization of DNA-functionalized gold nanoparticles, resulting in a disposable colorimetric biosensor. We present a new approach for the fabrication of dye sensitized TiO2 photodetectors by an inkjet printing technique-a non-contact digital, additive, no mask and no vacuum patterning method, ideal for cost efficient mass production. The developed biosensor was compared against a dye sensitized photodetector fabricated by the traditional {"}doctor blade{"} method. Detection of gold nanoparticle aggregation was possible for concentrations as low as 1.0 nM for the {"}doctor blade{"} system, and 1.5 nM for the inkjet printed photodetector. The demonstrated sensitivity limits of developed biosensors; are comparable to those of spectrophotometric techniques (1.0 nM). Our results show that a difference higher than 17% by traditional photodetector and 6% by inkjet printed in the photoresponses for the complementary and non-complementary gold nanoprobe assays could be attained for a specific DNA sequence from Mycobacterium tuberculosis, the etiologic agent of human tuberculosis. The decrease of costs associated with molecular diagnostic provided by a platform such as the one presented here may prove of paramount importance in developing countries. (C) 2009 Elsevier B.V. All rights reserved.

Gold nanoparticles functionalized with thiol-oligonucleotides are ideal platforms for detection of specific DNA sequences. Here we evaluate the effect of single base mismatches in hybridization efficiency according to the position of the mismatch, base pairing combination and thiol-oligonucleotide density in terms of specificity and efficiency of target recognition. Hybridization efficiency and single-nucleotide polymorphism discrimination at room temperature is maximized at a density of 83 +/- 4 thiol-oligonucleotides per 13.5 nm gold nanoparticle (24 pmol/cm(2)), and when the mismatch is localized at the 3'-end of the Au-nanoprobe, i.e. away from the gold nanoparticle surface. (C) 2010 Elsevier B.V. All rights reserved.

The present invention relates to a system and process for detection and/or qualitative and quantitative identification of the biological material, such as specific sequences of nucleic acids or proteins as antibodies, present in biological samples. The system is composed by one or more light sources (1) combined with one or more integrated optical photo sensors, or not, and various electronic components (4), necessary for obtaining/processing of the signal emitted by the metal nanoprobes functionalized with a solution of biological composite, as well as also a micro-controller and a microprocessor, fixed or portable. This photosensor structure is able to detect and to quantify the colour variations produced by metal nanoprobes, being this preferentially gold, functionalized by oligonucleotides complementary to specific DNA/RNA sequences, proteins, as for instance antibodies and/or antigens related with certain disease, or other sample or solution of biological composite, that are to be investigated. The detection and quantification process is based on the response of a photosensor, singular or integrated, based on thin film technology of amorphous, nanocrystalline or microcrystalline silicon and their alloys, as well as the new active ceramic semiconductors, amorphous and not amorphous.

The present invention relates to a system and process for detection and/or qualitative and quantitative identification of the biological material, such as specific sequences of nucleic acids or proteins as antibodies, present in biological samples. The system is composed by one or more light sources (1) combined with one or more integrated optical photo sensors, or not, and various electronic components (4), necessary for obtaining/ processing of the signal emitted by the metal nanoprobes functionalized with a solution of biological composite, as well as also a micro-controller and a microprocessor, fixed or portable. This photosensor structure is able to detect and to quantify the colour variations produced by metal nanoprobes, being this preferentially gold, functionalized by oligonucleotides complementary to specific DNA/RNA sequences, proteins, as for instance antibodies and/or antigens related with certain disease, or other sample or solution of biological composite, that are to be investigated. The detection and quantification process is based on the response of a photosensor, singular or integrated, based on thin film technology of amorphous, nanocrystalline or microcrystalline silicon and their alloys, as well as the new active ceramic semiconductors, amorphous and not amorphous.