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.

Coupling organic substrate removal to methane production in a fully biological microbial electrolysis cell

Microbial electrolysis cells (MECs) are an innovative and emerging technique based on the use of solid-state electrodes to stimulate microbial metabolism for wastewater treatment and simultaneous production of value-added compounds (such as methane). This research studied the performance of a two-chamber MEC in terms of organic matter oxidation (at the anode) and methane production (at the cathode). MEC‟s anode had been previously inoculated with an activated sludge, whereas the cathode chamber inoculum was an anaerobic sludge (containing methanogenic microorganisms). During the experimentation, the bioanode was continuously fed with synthetic solutions in anaerobic basal medium, at an organic load rate (OLR) of around 1 g L-1 d-1, referred to the chemical oxygen demand (COD). At the beginning (Run I), the feeding solution contained acetate and subsequently (Run II) it was replaced with a more complex solution containing soluble organic compounds other than acetate. For both conditions, the anode potential was controlled at -0.1 V vs. standard hydrogen electrode, by means of a potentiostat. During Run I, over 80% of the influent acetate was anaerobically oxidized at the anode, and the resulting electric current was recovered as methane at the cathode (with a cathode capture efficiency, CCE, accounting around 115 %). The average energy efficiency of the system (i.e., the energy captured into methane relative to the electrical energy input) under these conditions was over 170%. However, reactor‟s performance decreased over time during this run. Throughout Run II, a substrate oxidation over 60% (on COD basis) was observed. The electric current produced (57% of coulombic efficiency) was also recovered as methane, with a CCE of 90%. For this run the MEC‟s average energy efficiency accounted for almost 170 %. During all the experimentation, a very low biomass growth was observed at the anode whereas ammonium was transferred through the cationic membrane and concentrated at the cathode. Tracer experiments and scanning electron microscopy analyses were also carried out to gain a deeper insight into the reactor performance and also to investigate the possible reasons for partial loss of performance. In conclusion, this research suggests the great potential of MEC to successfully treat low-strength wastewaters, with high energy efficiency and very low sludge production.

Bacterial cellulose as a novel substrate for retinal pigment epithelium cells transplantation in age-related macular degeneration

Tese de Doutoramento em Engenharia Química e Biológica.

Assessment of the properties to characterize the interface between clay brick substrate and strengthening mortar

The behaviour of masonry elements under in-plane and out-of-plane loads can be improved through the application of strengthening systems based on reinforcing overlays. After strengthening, the transition region between the original substrate and the strengthening layer is especially stressed, and premature failure of the strengthened masonry is reached if insufficient interfacial capacity is assured. Therefore, the assessment of the mechanical behaviour of the interface is critical to the development of the masonry strengthening system based on the application of strengthening overlays. In this research a method for the characterization of the interface behaviour between two different materials, a polypropylene fibre reinforced mortar (PFRM) and a ceramic brick used for masonry construction is presented. Direct shear tests were carried out in couplet specimens. Due to the orthotropic nature of the bricks surface, the shear load was applied along three different directions in order to perform an overall estimation of the interface behaviour. The peak and residual shear stresses, as well as the failure modes, were obtained at different levels of the normal stress. Based on these experimental results constitutive laws were assessed for the simulation of the interface mechanical behaviour based on the Mohr and Mohr-Coulomb failure criteria.

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of Retinal Pigment Epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.52.0 swelling/mm3), high mechanical strength (2048 MPa) and non-pyrogenicity (2.12 EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored.

Lard as a suitable and cheap substrate for microbial lipids production by Yarrowia lipolytica

[Excerpt] Current agricultural and industrial practices have led to the generation of large amounts of various low-value or negative cost crude wastes, which are difficult and economically notattractive to treat and valorize. One important example of waste generation is animal fat, commonly found in tanning process and slaughterhouses. These wastes, in which the lipids are often the main and most problematic components, are not currently used effectively and there are almost no application methods to recover the respective value. (...)

The relation of the concentration of macronutrients in the substrate and in the foliage to cell wall thickness and cellulose concentration in the xylem of slash pine (Pinus elliotti)

Sand culture experiments, using a sub-irrigation technique, were installed in order to find out the effects of the macronutrients N, P, K, Ca, Mg and S on growth, aspect, mineral composition, length of fibers, thickness of cell wall and cellulose concentration in slash pine. The aim was to obtain, under controlled conditions, basic information which could eventually lead to practical means designed to increase the rate of growth and to make of slash pine a richer source of cellulose. Nitrogen, Phosphorus, Potassium Experiment A 3 x 3 x 3 factorial design with two replicates was used. Nitrogen was supplied initially at the levels of 25, 50 and 100 ppm; phosphorus was given at the rates of 5, 10 and 20 ppm; potassium was supplied at the rates of 25, 50 and 100 ppm; six months after the experiment was started the first level for each element was dropped to zero. Others macro and all micronutrients were supplied at uniform rates. Fifteen hours of illumination per day were provided. The experimental technique for growing the slash pine seedlings proved quite satisfactory. Symptoms of deficiency of nitrogen, phosphorus and potassium were observed, described and recorded in photographs and water colors. These informations will help to identify abnormalities which may appear under field conditions. Chemical analysis of the several plant parts, on the other hand, give a valuable means to assess the nutritional status of slash pine, thus confirming when needed, the visual diagnosis. The correctness of manurial pratices, on the other hand, can be judged with the help of the analytical data tabulated. Under the experimental conditions nitrogen caused the highest increases on growth, as measured by increments in height and dry weights, whereas the effects of phosphorus and potassium were less marked. Cellulose concentration was not significantly affected by the treatments used. Higher levels of N seemed to decrease both length of fiber elements and the thickness of cell wall. The effects of P and K were not well defined. Calcium, Magnesium, Sulfur Experiment A 3 x 3 x 3 factorial design with two replicates was used. Calcium was supplied initially at the levels of 12.5, 25 and 50 ppm; magnesium and sulfur were given at the rates of 6, 12.5 and 25 ppm. Other macro and micronutrients were supplied at uniform rates, common to all treatments. Three months after starting the experiment the first level for each element was dropped to zero. Symptoms of deficiency of calcium, magnesium and sulfur were observed, described and recorded as in the case of the previous experiment. Chemical analysis were made, both for mineral content and cellulose concentration. Length of fibers and thickness of cell wall were measured. Both calcium and magnesium increase height, sulfur failing to give significant response. Dry weight was beneficially affected by calcium and sulfur. The levels of calcium, magnesium and sulfur in the needles associated with deficiency and maximum growth are comparable with those found in the literature. Cellulose concentration increased when the level of sulfur in the substrate was raised. The thickness of cell wall was negatively affected by the treatments; no effect was observed with regards to length of fibers.

Finite element simulation of an impinging liquid droplet on a hot solid substrate

Magdeburg, Univ., Fak. für Mathematik, Diss., 2014

Role of divalent cations, pH, cytoskeleton componentes and surface charge on the adhesion of Trichomonas vaginalis to a polystyrene substrate

The process of adhesion of three different strains of Trichomonas vaginalis to a polystyrene substrate was analysed. The process of adhesion was dependent on the time of incubation and the pH of the phosphate-buffered solution (PBS) in which the parasites were suspended. The highest indices of adhesion were observed after an incubation time of 60 min at pH 6.6. The adhesion index increased when the parasites were incubated in the presence of culture media or when Ca++ or Mg++ was added to the PBS solution, whereas cytochalasin B, trypsin or neuraminidase reduced adhesion. Incubation of the parasites in the presence of poly-L-lysine facilitated the process of adhesion. Incubation of the parasites or polystyrene beads in the presence of poly-L-lysine led to important changes in their surface charge.

MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.

Tailed Duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing.

Purpose of the Study: To elucidate the mechanism of homologous recombination and double-strand break repair mediated by the eukaryotic recombination pin, Rad51.