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.

Aims: Our aim is to explore whether gold nanoparticles (AuNPs) functionalized with a carboxylated polyethylene glycol (PEG) and protamine (AuNP@PEG@Prot) can modulate - enhance or restrain - DNA condensation, altering DNA conformation and inducing structural changes. Understanding how these nanoconjugates modulate DNA structure, size and shape of DNA condensates, and enable control over the resulting 3D structures is of major biological and therapeutic importance. Materials & methods: Citrate-AuNPs were covered with a dense layer of a hetero-functional octa(ethylene glycol) (SH-EG(8)-COOH). Conjugation of protamine to the AuNP@PEG was achieved by taking advantage of the carboxylated surface previously generated on the surface of the NP and the remaining amino groups from the protamine, using carbodiimide and N-hydroxysulfosuccinimide coupling reactions. Results & conclusion: AuNP@PEG@Prot modulates the structure and topology of DNA, not only for condensation, but also for decondensation, via formation of higher quantities of dimers and multimers, when compared with AuNP@PEG and free protamine.

The surface chemistry of gold nanoparticles (AuNPs) plays a critical role in the self-assembly of thiolated molecules and in retaining the biological function of the conjugated biomolecules. According to the well-established gold-thiol interaction the undefined ionic species on citrate-reduced gold nanoparticle surface can be replaced with a self-assembled monolayer of certain thiolate derivatives and other biomolecules. Understanding the effect of such derivatives in the functionalization of several types of biomolecules, such as PEGs, peptides or nucleic acids, has become a significant challenge. Here, an approach to attach specific biomolecules to the AuNPs (∼14 nm) surface is presented together with a study of their effect in the functionalization with other specific derivatives. The effect of biofunctional spacers such as thiolated poly(ethylene glycol) (PEG) chains and a positive peptide, TAT, in dsRNA loading on AuNPs is reported. Based on the obtained data, we hypothesize that loading of oligonucleotides onto the AuNP surface may be controlled by ionic and weak interactions positioning the entry of the oligo through the PEG layer. We demonstrate that there is a synergistic effect of the TAT peptide and PEG chains with specific functional groups on the enhancement of dsRNA loading onto AuNPs.

Nanotechnology provides new tools for gene expression analysis that allow for sensitive and specific characterization of prognostic signatures related to cancer. Cancer is a multigenic complex disease where multiple gene loci contribute to the phenotype. The ability to simultaneously monitor differential expression originating from each locus allows for a more accurate indication of degree of cancerous activity than either locus alone. Metal nanoparticles have been widely used as labels for in vitro identification and quantification of target sequences. Here we describe the synthesis of nanoparticles with different noble metal compositions in an alloy format that are then functionalized with thiol-modified ssDNA (nanoprobes). We also show how to use such nanoprobes in a non-cross-linking colorimetric method for the direct detection and quantification of specific mRNA targets, without the need for enzymatic amplification or reverse transcription steps. The different metals in the alloy provide for distinct absorption spectra due to their characteristic plasmon resonance peaks. The color multiplexing allows for simultaneous identification of several different mRNA targets involved in cancer development. Comparison of the absorption spectra of the nanoprobes mixtures taken before and after induced aggregation of metal nanoparticles allows to both identify and quantify each mRNA target. We describe the use of gold and gold:silver-alloy nanoprobes for the development of the non-cross-linking method to detect a specific BCR-ABL fusion gene (e.g., e1a2 and e14a2) mRNA target associated with chronic myeloid leukemia (CML) using 10 ng μL -1 of unamplified total human RNA. This simple methodology takes less than 50 min to complete after total RNA extraction with comparable specificity and sensitivity to the more commonly used methods.

A strategy for the development of novel antimicrobials is to combine the stability and pleiotropic effects of inorganic compounds with the specificity and efficiency of organic compounds, such as antibiotics. Here we report on the use of gold:silver-alloy (Au:Ag-alloy) nanoparticles, obtained via a single-step citrate co-reduction method, combined to conventional antibiotics to enhance their antimicrobial effect on bacteria. Addition of the alloy nanoparticles considerably decreased the dose of antibiotic necessary to show antimicrobial effect, both for bacterial cells growing in rich medium in suspension and for bacterial cells resting in a physiological buffer on a humid cellulose surface. The observed effect was more pronounced than the sum of the individual effects of the nanoparticles and antibiotic. We demonstrate the enhancement effect of Au:Ag-alloy nanoparticles with a size distribution of 32.5±7.5nm mean diameter on the antimicrobial effect of (i) kanamycin onEscherichia coli(Gram-negative bacterium), and (ii) a β-lactam antibiotic on both a sensitive and resistant strain ofStaphylococcus aureus(Gram-positive bacterium). Together, these results may pave the way for the combined use of nanoparticle–antibiotic conjugates towards decreasing antibiotic resistance currently observed for certain bacteria and conventional antibiotics.

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.

Several ultrasound-based platforms for DNA sample preparation were evaluated in terms of effective fragmentation of DNA (plasmid and genomic DNA)-ultrasonic probe, sonoreactor, ultrasonic bath and the newest Vialtweeter device. The sonoreactor showed the best efficiency of DNA fragmentation while simultaneously assuring no cross-contamination of samples, and was considered the best ultrasonic tool to achieve effective fragmentation of DNA at high-throughput and avoid sample overheating. Several operation variables were studied-ultrasonication time and amplitude, DNA concentration, sample volume and sample pre-treatment-that allowed optimisation of a sonoreactor-based strategy for effective DNA fragmentation. Optimal operating conditions to achieve DNA fragmentation were set to 100% ultrasonic amplitude, 100 μL sample volume, 8 min ultrasonic treatment (2 min/sample) for a DNA concentration of 100 μg mL-1. The proposed ultrasonication strategy can be easily implemented in any laboratory setup, providing fast, simple and reliable means for effective DNA sample preparation when fragmentation is critical for downstream molecular detection and diagnostics protocols.

This paper describes a novel colorimetric method for detection of nucleic acid targets in a homogeneous format with improved sensitivity by means of a system based on the combination of a tunable monochromatic light source and an amorphous/nanocrystalline silicon photodetector that detects color and light intensity changes undergone by samples/assays containing tailored gold nanoparticles probes. This new low cost, portable, fast and simple optoelectronic platform, with the possibility to be re-used, permits detection of at least 400 fentomole of specific DNA sequences without target or signal amplification and was applied to the rapid detection of human pathogens in large variety of clinical samples such as Mycobacterium tuberculosis.

Amorphous/nanocrystalline silicon pi'ii'n devices fabricated on micromachined glass substrates are integrated with oligonucleotide-derivatized gold nanoparticles for a colorimetric detection method. The method enables the specific detection and quantification of unamplified nucleic acid sequences (DNA and RNA) without the need to functionalize the glass surface, allowing for resolution of single nucleotide differences between DNA and RNA sequences-single nucleotide polymorphism and mutation detection. The detector's substrate is glass and the sample is directly applied on the back side of the biosensor, ensuring a direct optical coupling of the assays with a concomitant maximum photon capture and the possibility to reuse the sensor. (c) 2007 American Institute of Physics.

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.