Gold nanoparticle-DNA conjugates for oligonucleotide vectorization towards gene silencing

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

mRNA genotyping by gold nanoprobes

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Gold nanoparticle to antibody conjugates for diagnosis applications: molecular interactions and immunoassay development

Thesis for the Master degree in Structural and Functional Biochemistry

Detection of Arah1 (a major peanut allergen) in food using an electrochemical gold nanoparticle-coated screen-printed immunosensor

A gold nanoparticle-coated screen-printed carbon electrode was used as the transducer in the development of an electrochemical immunosensor for Ara h 1 (a major peanut allergen) detection in food samples. Gold nanoparticles (average diameter=32 nm) were electrochemically generated on the surface of screen-printed carbon electrodes. Two monoclonal antibodies were used in a sandwich-type immunoassay and the antibody–antigen interaction was electrochemically detected through stripping analysis of enzymatically (using alkaline phosphatase) deposited silver. The total time of the optimized immunoassay was 3 h 50 min. The developed immunosensor allowed the quantification of Ara h 1 between 12.6 and 2000 ng/ml, with a limit of detection of 3.8 ng/ml, and provided precise (RSD <8.7%) and accurate (recovery >96.6%) results. The immunosensor was successfully applied to the analysis of complex food matrices (cookies and chocolate), being able to detect Ara h 1 in samples containing 0.1% of peanut.

Dynamic stability metrics for the container loading problem

The Container Loading Problem (CLP) literature has traditionally evaluated the dynamic stability of cargo by applying two metrics to box arrangements: the mean number of boxes supporting the items excluding those placed directly on the floor (M1) and the percentage of boxes with insufficient lateral support (M2). However, these metrics, that aim to be proxies for cargo stability during transportation, fail to translate real-world cargo conditions of dynamic stability. In this paper two new performance indicators are proposed to evaluate the dynamic stability of cargo arrangements: the number of fallen boxes (NFB) and the number of boxes within the Damage Boundary Curve fragility test (NB_DBC). Using 1500 solutions for well-known problem instances found in the literature, these new performance indicators are evaluated using a physics simulation tool (StableCargo), replacing the real-world transportation by a truck with a simulation of the dynamic behaviour of container loading arrangements. Two new dynamic stability metrics that can be integrated within any container loading algorithm are also proposed. The metrics are analytical models of the proposed stability performance indicators, computed by multiple linear regression. Pearson’s r correlation coefficient was used as an evaluation parameter for the performance of the models. The extensive computational results show that the proposed metrics are better proxies for dynamic stability in the CLP than the previous widely used metrics.

Active bionanoconjugates of laccase and gold nanoparticles: kinetic and structural studies

Thesis for the master degree in Structural and Functional Biochemistry

Using gold nanoparticles in protein crystallography: studies in crystal growth and derivatization

Dissertation for the Master Degree in Structural and Functional Biochemistry

Liver function evaluation in leptospirosis with colloidal gold 1 9 8 Au

Eight patients with leptospirosis were studied with colloidal gold 1 9 8 Au. The radiocolloidal hepatic distribution was altered, presenting a non-homogeneous tiver concentration in seven cases, and a minute to moderate splenic visualization in five. Two patients presented doubtful splenic image, and one seemed to be normal. Liver scanning with colloidal gold 1 9 8 Au is demonstra ted to be a good liver function test.

Long-term behaviour of railway transitions under dynamic loading application to soft soil sites

Dissertação para obtenção do Grau de Doutor em Engenharia Civil

Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Cancer theranostics: multifunctional gold nanoparticles for diagnostics and therapy

Doctorate in Biology, Specialty in Biotechnology

Mercury exposure and malaria prevalence among gold miners in Pará, Brazil

Economic development, including resource extraction, can cause toxic exposures that interact with endemic infectious diseases. Mercury is an immunotoxic metal used in the amalgamation of gold, resulting in both occupational exposures and environmental pollution. A cross-sectional medical survey was conducted in 1997 on 135 garimpeiros in Para, Brazil, because of their risks of both mercury exposure and malaria transmission. Mean levels of blood and urine mercury were well above non-exposed background levels. Twenty-six subjects had malaria parasitemia: Health symptoms consistent with mercury exposure were reported, but neither symptoms nor signs correlated with mercury levels in blood or urine. We did not find a dose response relationship between mercury exposure and likelihood of prevalent malaria infection, but there was a possible reduction in acquisition of immunity that may be associated with conditions in gold mining, including mercury exposure.

Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays

Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use. Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area. Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art. Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation. Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.

Gold nanoparticles for plasmid delivery

Gene therapy presents an ideal strategy for the treatment of genetic as well as acquired diseases, such as cancer and typically involves the insertion of a functioning gene into cells to correct a cellular dysfunction or to provide a new cellular function. Gene delivery vectors are based in two models: viral and non-viral. Viral vectors have high transfection efficiency but their major barrier is immunogenicity. Since the non-viral vectors have no immunogenicity, these have been widely studied. Gold nanoparticles have been proposed as optimal delivery systems of genetic material, due their small size, high surface-to-volume ratio and the ability to be functionalized with multiple molecules. In the present work, an AuNP-based formulation was developed to deliver a plasmid in a colorectal cancer cell line, containing as reporter gene the gene encoding to EGFP. The delivery system resulted from the functionalization of 14 nm AuNP with a PEG layer (4300114 PEG chains/AuNP), which increases stability and biocompatibility of AuNPs; quaternary ammonium groups which provide positive charges that allow electrostatic binding of plasmid, which is considered the therapeutic agent to be transported into cells. The system developed was characterized by UV-vis spectroscopy, DLS, TEM and by electrophoretic mobility, yielding a formulation with 113.5 nm.Transfection efficiency of the formulation developed was evaluated through PCR and through EGFP expression by fluorescence microscopy and fluorescence spectroscopy. The internalization was observed 3h post transfection; however a low level of EGFP expression was achieved. After 24h of incubation, EGFP expression increases just 3 times compared to non-transfected cells. The commercial system (Lipofectamine) expressed EGFP 5 times more than the system developed AuNP@PEG@R4N+@pEGFP. This difference could be related to lower translocation to the nucleus.

Gold nanoparticulate systems for new anticancer compounds delivery

My master studies have resulted in the following publication: Martins P, Rosa D, Fernandes AR, Baptista PV. 2014. Nanoparticle Drug Delivery Systems: Recent Patents and Applications in Nanomedicine. Recent Patents in Nanomedicine. 3(2):105-118.