Nanoparticles have been making their way in biomedical applications and personalized medicine, allowing for the coupling of diagnostics and therapeutics into a single nanomaterial-nanotheranostics. Gold nanoparticles, in particular, have unique features that make them excellent nanomaterials for theranostics, enabling the integration of targeting, imaging and therapeutics in a single platform, with proven applicability in the management of heterogeneous diseases, such as cancer. In this review, we focus on gold nanoparticle-based theranostics at the lab bench, through pre-clinical and clinical stages. With few products facing clinical trials, much remains to be done to effectively assess the real benefits of nanotheranostics at the clinical level. Hence, we also discuss the efforts currently being made to translate nanotheranostics into the market, as well as their commercial impact.

Background: Gold nanoparticles have been widely employed for biosensing purposes with remarkable efficacy for DNA detection. Amongst the proposed systems, colorimetric strategies based on the remarkable optical properties have provided for simple yet effective sequence discrimination with potential for molecular diagnostics at point of need. These systems may also been used for parallel detection of several targets to provide additional information on diagnostics of pathogens.Results: For the first time, we demonstrate that a single Au-nanoprobe may provide for detection of two distinct targets (pathogens) allowing colorimetric multi-target detection. We demonstrate this concept by using one single gold-nanoprobe capable to detect members of the Mycobacterium tuberculosis complex and Plasmodium sp., the etiologic agents of tuberculosis and malaria, respectively. Following characterisation, the developed gold-nanoprobe allowed detection of either target in individual samples or in samples containing both DNA species with the same efficacy.Conclusions: Using one single probe via the non-cross-linking colorimetric methodology it is possible to identify multiple targets in one sample in one reaction. This proof-of-concept approach may easily be integrated into sensing platforms allowing for fast and simple multiplexing of Au-nanoprobe based detection at point-of-need.

The design and preparation of highly efficient drug delivery platforms using green methodologies is at the forefront of nanotherapeutics research. POxylated polyurea dendrimers are efficiently synthesized using a supercritical-assisted polymerization in carbon dioxide. These fluorescent, pH-responsive and water-soluble core-shell smart nanocarriers show low toxicity in terms of cell viability and absence of glutathione depletion, two of the major side effect limitations of current vectors. The materials are also found to act as good transfection agents, through a mechanism involving an endosomal pathway, being able to reduce 100-fold the IC50 of paclitaxel.

Tuberculosis, still one of the leading human infectious diseases, reported 8.7 million new cases in 2011 alone. Also, the increasing rate of multidrug-resistant tuberculosis (MDRTB) and its treatment difficulties pose a serious public health threat especially in developing countries. Resistance to isoniazid and rifampicin, first line antibiotics, is commonly associated with point mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis complex (MTBC). Therefore, the development of cheap, fast and simple molecular methods to assess susceptibility profiles would have a huge impact in the capacity of early diagnosis and treatment of MDRTB. Gold nanoparticles functionalized with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for MTBC and single base mutations associated with antibiotic resistance, namely the characterization of the three most relevant codons in rpoB gene associated to rifampicin resistance. Here we extend the Au-nanoprobe approach towards discriminating specific mutations within inhA and rpoB genes in PCR amplified DNA from isolates. Using a multiplex PCR reaction for these two genes, it is possible to assess both loci in parallel, and extend the potential of the Au-nanoprobe method to MDRTB molecular characterization with special application in the most frequent Portuguese genotypes. (C) 2014 Elsevier Ltd. All rights reserved.

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.

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.

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.

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.

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.

Tuberculosis remains one of the most serious infectious diseases worldwide requiring new tools to circumvent current molecular diagnostics limitations. Nanodiagnostics, i.e. nanotechnology based diagnostics, may do just that by decreasing the time needed for the molecular characterisation of the infecting agent, and allowing for miniaturisation and portability for point-of-need adapted to remote regions without suitable lab equipment.

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.

Tuberculosis (TB) is one of the leading causes of infection in humans, causing high morbility and mortality all over the world. The rate of new cases of multidrug resistant tuberculosis (MDRTB) continues to increase, and since these infections are very difficult to manage, they constitute a serious health problem. In most cases, drug resistance in Mycobacterium tuberculosis has been related to mutations in several loci within the pathogen's genome. The development of fast, cheap and simple screening methodologies would be of paramount relevance for the early detection of these mutations, essential for the timely and effective diagnosis and management of MDRTB patients. The use of gold nanoparticles derivatized with thiol-modified oligonucleotides (Au-nanoprobes) has led to new approaches in molecular diagnostics. Based on the differential non-cross-linking aggregation of Au-nanoprobes, we were able to develop a colorimetric method for the detection of specific sequences and to apply this approach to pathogen identification and single base mutations/single nucleotide polymorphisms (SNP) discrimination. Here we report on the development of Au-nanoprobes for the specific identification of SNPs within the beta subunit of the RNA polymerase (rpoB locus), responsible for resistance to rifampicin in over 95% of rifampicin resistant M. tuberculosis strains.

We recently reported on the use of caged nucleotides to attain full control of enzymatic polymerization of RNA solely by light. In the absence of light no RNA formation was possible due to the efficient caging by the coumarin moiety; after irradiation, caged ATP was released with quantitative precision and RNA polymerization was resumed. As photolabile protecting group [7-(diethylamino) coumarin-4-yl]methyl] (DEACM) was used due to its high absorbance in the visible region of the spectrum, fast deprotection kinetics and absence of radical intermediates. However, the 7-diethylamino-4-hydroxymethylcoumarin photo-product (DEACM-OH) was shown to inhibit the transcription reaction for concentrations higher than 30 μM [5]. This inhibition has been associated with poor water solubility, which is commonly dealt with via cumbersome chemical modifications of the protecting moiety. To overcome inhibition, we evaluated the use of molecular scavengers to sequester DEACM-OH formed after irradiation. Determination of association constants of coumarin with β-cyclodextrins allowed the assessment of its capability to remove free coumarin molecules from solution. The influence of β-cyclodextrin in transcription reaction was also assessed. Results show that β-cyclodextrin can be successfully used as scavenger as it increases the DEACM-OH threshold concentration for inhibition, amplifying the efficiency of light controlled in vitro transcription.

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.

There are numerous reports of coumarin ester derivatives, in particular phosphate esters, as photocleavable cages in biological systems. Despite the comprehensive analysis of the photocleavage mechanism, studies of 4-methylcoumarin caged phosphates and/or nucleotides were always performed at constant pH. In this work, we present the study of the pH effect on the photochemistry of (7-diethylaminocoumarin-4-yl)methyl phosphate (DEACM-P). Fluorescence and photocleavage quantum yields, as well as the fluorescence decay times were measured as a function of the pH. It was found that the pH produces significant changes in the overall photochemical quantum yield of DEACM-P, and the observed changes are complementary to those obtained from the fluorescence quantum yield. Deprotonation of DEACM-HPO4 - to yield DEACM-PO4 2-, produces a decrease in the photochemical quantum yield (from 0.0045 to 0.0003) and an increase in the fluorescence quantum yield (from 0.072 to 0.092). Moreover, from the analysis of the decay times, we have also found that hydroxyl ion is not only relevant, but it is mechanistically involved in the photoreaction of DEACM-HPO4 -.

O presente invento relaciona-se com um método para controlo de reac{\c c}ões enzimáticas de síntese de ácidos nucleicos, recorrendo a nucleótidos funcionalizados com derivados de 4-metilcumarinas (1) protectores e fotolábeis. Quando ligado aos nucleótidos (2), o grupo cumarinico (3) impede que estes sejam utilizados como substrato por parte das enzimas, impossibilitando a ocorrência de reac{\c c}ão. Através de irradia{\c c}ão com radia{\c c}ão electromagnética, o grupo cumarinico é libertado, ficando o nucleótido disponível para a reac{\c c}ão. Desta forma, as reac{\c c}ões enzimáticas de síntese de ácidos nucleicos podem ser controladas através da luz.

FRET can be used as a strategy to assign different simultaneous events in the same sample but {"}cross-talk{"} problems are a limitation. Here we present a contribution for the better understanding of the {"}cross-talk{"} in FRET experiments that include several pairs in the same sample. Using oligonucleotide probes labeled with fluorescent dyes which can be selectively excited at a specific wavelength, and using target oligonucleotides tagged with a fluorescent dye with specific characteristics that allow only it to emit light upon selective excitation of a specific probe by energy transfer (FRET), we aim to identify the exact probe-target hybridized pair. When using three donors to probe the presence of complementary targets, only 20% of possible donor/acceptor combinations give straightforward signals readily identifiable with the sample composition, while in the remaining cases severe cross-excitation prevents the direct identification of the sample composition. To correctly resolve the samples identity, we developed a theoretical model that enables the unequivocal attribution of a sample composition to a given set of fluorescence signals, in the presence of three donors.

O presente invento relaciona-se com um novo sistema e processo para detec{\c c}ão e/ou identifica{\c c}ão qualitativa e quantitativa de matéria biológica, tais como sequências específicas de ácidos nucleicos ou proteínas, como anticorpos, presentes em amostras biológicas. O sistema é constituído por uma ou mais fontes luminosas combinadas, com um ou mais fotossensores ópticos integrados, ou não, e componentes electrónicos vários, necessários para obten{\c c}ão/condicionamento do sinal emitido por nanossondas de metal funcionalizadas com a solu{\c c}ão de composto biológico, bem como ainda um micro-controlador e um microprocessador, portável ou fixo. Este fotossensor é capaz de detectar e quantificar as diferen{\c c}as colorimétricas produzidas por nanossondas de metal, sendo este preferencialmente o ouro, funcionalizadas por oligonucleotídeos complementares às sequências específicas de ADN/ARN, proteínas, como por exemplo anticorpos e/ou antigénios relacionados com determinada doen{\c c}a, ou outra amostra ou solu{\c c}ão de composto biológico, que se pretende pesquisar. O processo de detec{\c c}ão e quantifica{\c c}ão baseia-se na resposta de um fotossensor, singular ou integrado, baseado na tecnologia de filmes finos de sílicio amorfo, nanocristalino ou mícrocristalino, e suas ligas, e também nos novos cerâmicos semicondutores activos, amorfos e não morfos. O referido sistema e processo de detec{\c c}ão e/ou identifica{\c c}ão de matéria biológica tem aplica{\c c}ão na biotecnologia, incluindo a biomedicina.

The impact of advances in nanotechnology is particularly relevant in biodiagnostics, where nanoparticle-based assays have been developed for specific detection of bioanalytes of clinical interest. Gold nanoparticles show easily tuned physical properties, including unique optical properties, robustness, and high surface areas, making them ideal candidates for developing biomarker platforms. Modulation of these physicochemical properties can be easily achieved by adequate synthetic strategies and give gold nanoparticles advantages over conventional detection methods currently used in clinical diagnostics. The surface of gold nanoparticles can be tailored by ligand functionalization to selectively bind biomarkers. Thiol-linking of DNA and chemical functionalization of gold nanoparticles for specific protein/antibody binding are the most common approaches. Simple and inexpensive methods based on these bio-nanoprobes were initially applied for detection of specific DNA sequences and are presently being expanded to clinical diagnosis.

We describe the use of ATP caged with [7-(diethylamino)coumarin-4-yl]methyl (DEACM) for light-controlled in vitro transcription reactions. Polymerization is blocked when DEACM is bonded to the gamma phosphate group of the ATP molecule. Controlled light irradiation releases ATP and transcription is initiated. In order to provide full control over the process, conditions involved in substrate release, nucleotide availability after release and the effect of the released coumarin in RNA polymerization were assessed in further detail. Together, our data provide the first direct evidence of control over enzymatic polymerization of nucleic acids through light. This approach may provide researchers with a unique tool for the study of biological processes at a molecular level.

Thiol-linked DNA-gold nanoparticles were used in a novel colorimetric method to detect the presence of specific mRNA from a total RNA extract of yeast cells. The method allowed detection of expression of the FSY1 gene that encodes a specific fructose/H+ symporter in Saccharomyces bayanus PYCC 4565. FSY1 is strongly expressed when the yeast is grown in fructose as the sole carbon source, while cells cultivated in glucose as the sole carbon source repress gene expression. The presence of FSY1 mRNA is detected based on color change of a sample containing total RNA extracted from the organism and gold nanoparticles derivatized with a 15-mer of complementary single stranded DNA upon addition of NaCl. If FSY1 mRNA is present, the solution remains pink, changing to blue-purple in the absence of FSY1 mRNA. Direct detection of specific expression was possible from only 0.3 microg of unamplified total RNA without any further enhancement. This novel method is inexpensive, very easy to perform as no amplification or signal enhancement steps are necessary and takes less than 15 min to develop after total RNA extraction. No temperature control is necessary and color change can be easily detected visually.

We present a high-resolution bacterial contig map of 3.4 Mb of genomic DNA in human chromosome 21q11-q21, encompassing the region of elevated disomic homozygosity in Down Syndrome-associated abnormal myelopoiesis and leukemia, as well as the markers, which has shown a strong association with Alzheimer's Disease that has never been explained. The map contains 89 overlapping PACs, BACs, or cosmids in three contigs (850, 850, and 1500 kb) with two gaps (one of 140-210 kb and the second < 5 kb). To date, eight transcribed sequences derived by cDNA selection, exon trapping, and/or global EST sequencing have been positioned onto the map, and the only two genes so far mapped to this cytogenetic region, STCH and RIP140 have been precisely localized. This work converts a further 10% of chromosome 21q into a high-resolution bacterial contig map, which will be the physical basis for the long-range sequencing of this region. The map will also enable positional derivation of new transcribed sequences, as well as new polymorphic probes, that will help in elucidation of the role the genes in this region may play in abnormal myelopoiesis and leukemia associated with trisomy 21 and Alzheimer's Disease.