In the last decade the use of nanomaterials has been having a great impact in biosensing. In particular, the unique properties of noble metal nanoparticles have allowed for the development of new biosensing platforms with enhanced capabilities in the specific detection of bioanalytes. Noble metal nanoparticles show unique physicochemical properties (such as ease of functionalization via simple chemistry and high surface-to-volume ratios) that allied with their unique spectral and optical properties have prompted the development of a plethora of biosensing platforms. Additionally, they also provide an additional or enhanced layer of application for commonly used techniques, such as fluorescence, infrared and Raman spectroscopy. Herein we review the use of noble metal nanoparticles for biosensing strategies—from synthesis and functionalization to integration in molecular diagnostics platforms, with special focus on those that have made their way into the diagnostics laboratory.

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis . These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices.

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.

Nucleic acid amplification technologies (NAATs) have become fundamental tools in molecular diagnostics, due to their ability to detect small amounts of target molecules. Since its development, Polymerase Chain Reaction (PCR) has been the most exploited method, being stablished as the “gold standard” technique for DNA amplification. However, the requirement for different working temperatures leads to the need of a thermocycler machine or complex thermal apparatus, which have been preventing its application in novel integrated devices for single workflow and high throughput analysis. Conversely, isothermal amplification methods have been gaining attention, especially for point-of-care diagnosis and applications. These non-PCR based methods have been developed by mimicking the in vivo amplification mechanisms, while performing the amplification with high sensitivity, selectivity and allowing for high-throughput analysis. These favorable capabilities have pushed forward the implementation and commercialization of several platforms that exploit isothermal amplification methods, mostly against virus, bacteria and other pathogens in water, food, environmental and clinical samples. Nevertheless, the future of isothermal amplification methods is still dependent on achieving technical maturity and broader commercialization of enzymes and reagents.