Factorization of elliptic boundary value problems by invariant embedding and application to overdetermined problems

Dissertação para obtenção do Grau de Doutor em Matemática

Study on solubility of pharmaceutical compounds in ionic liquids

Dissertation to obtain the degree of master in Chemical and Biochemical Engineering

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.

Engineering of metal oxide nanoparticles for application in electrochemical devices

The growing demand for materials and devices with new functionalities led to the increased inter-est in the field of nanomaterials and nanotechnologies. Nanoparticles, not only present a reduced size as well as high reactivity, which allows the development of electronic and electrochemical devices with exclusive properties, when compared with thin films. This dissertation aims to explore the development of several nanostructured metal oxides by sol-vothermal synthesis and its application in different electrochemical devices. Within this broad theme, this study has a specific number of objectives: a) research of the influence of the synthesis parameters to the structure and morphology of the nanoparticles; b) improvement of the perfor-mance of the electrochromic devices with the application of the nanoparticles as electrode; c) application of the nanoparticles as probes to sensing devices; and d) production of solution-pro-cessed transistors with a nanostructured metal oxide semiconductor. Regarding the results, several conclusions can be exposed. Solvothermal synthesis shows to be a very versatile method to control the growth and morphology of the nanoparticles. The electrochromic device performance is influenced by the different structures and morphologies of WO3 nanoparticles, mainly due to the surface area and conductivity of the materials. The dep-osition of the electrochromic layer by inkjet printing allows the patterning of the electrodes without wasting material and without any additional steps. Nanostructured WO3 probes were produced by electrodeposition and drop casting and applied as pH sensor and biosensor, respectively. The good performance and sensitivity of the devices is explained by the high number of electrochemical reactions occurring at the surface of the na-noparticles. GIZO nanoparticles were deposited by spin coating and used in electrolyte-gated transistors, which promotes a good interface between the semiconductor and the dielectric. The produced transistors work at low potential and with improved ON-OFF current ratio, up to 6 orders of mag-nitude. To summarize, the low temperatures used in the production of the devices are compatible with flexible substrates and additionally, the low cost of the techniques involved can be adapted for disposable devices.

Healthcare- Associated infections: An analysis of costs in a Portuguese Hospital

Hospital-acquired infections (HAIs) delay healing, prolong Hospital stay, and increase both Hospital costs and risk of death. This study aims to estimate the extra length of stay and mortality rate attributable to each of the following HAIs: wound infection (WI); bloodstream infection (BSI); urinary infections (UI); and Hospital-acquired pneumonia (HAP). The study population consisted of patients discharged in CHLC in 2014. Data was collected to identify demographic information, surgical operations, development of HAIs and its outputs. The study used regressions and a matched strategy to compare cases (infected) and controls (uninfected). The matching criteria were: age, sex, week and type of admission, number of admissions, major diagnostic category and type of discharge. When compared to matched controls, cases with HAI had a higher mortality rate and greater length of stay. WI related to hip or knee surgery, increased mortality rate by 27.27% and the length of stay by 74.97 days. WI due to colorectal surgery caused an extra mortality rate of 10.69% and an excess length of stay of 20.23 days. BSI increased Hospital stay by 28.80 days and mortality rate by 32.27%. UI caused an average additional length of stay of 19.66 days and risk of death of 12.85%. HAP resulted in an extra Hospital stay of 25.06 days and mortality rate of 24.71%. This study confirms the results of the previous literature that patients experiencing HAIs incur in an excess of mortality rates and Hospital stay, and, overall, it presents worse results comparing with other countries.

Biotransformation/separation routes to obtain pure enantiomers

The objective of the work presented in this thesis was the development of an innovative approach for the separation of enantiomers of secondary alcohols, combining the use of an ionic liquid (IL) - both as solvent for conducting enzymatic kinetic resolution and as acylating agent - with the use of carbon dioxide (CO2) as solvent for extraction. Menthol was selected for testing this reaction/separation approach due to the increasing demand for this substance, which is widely used in the pharmaceutical, cosmetics and food industries. With a view to using an ionic ester as acylating agent, whose conversion led to the release of ethanol, and due to the need to remove this alcohol so as to drive reaction equilibrium forward, a phase equilibrium study was conducted for the ehtanol/(±)-menthol/CO2 system, at pressures between 8 and 10 MPa and temperatures between 40 and 50 oC. It was found that CO2 is more selective towards ethanol, especially at the lowest pressure and highest temperature tested, leading to separation factors in the range 1.6-7.6. The pressure-temperature-composition data obtained were correlated with the Peng-Robinson equation of state and the Mathias-Klotz-Prausnitz mixing rule. The model fit the experimental results well, with an average absolute deviation (AAD) of 3.7 %. The resolution of racemic menthol was studied using two lipases, namely lipase from Candida rugosa (CRL) and immobilized lipase B from Candida antarctica (CALB), and two ionic acylating esters. No reaction was detected in either case. (R,S)-1-phenylethanol was used next, and it was found that with CRL low, nonselective, conversion of the alcohol took place, whereas CALB led to an enantiomeric excess (ee) of the substrate of 95%, at 30% conversion. Other acylating agents were tested for the resolution of (±)-menthol, namely vinyl esters and acid anhydrides, using several lipases and varying other parameters that affect conversion and enantioselectivity, such as substrate concentration, solvent and temperature. One such acylating agent was propionic anhydride. It was thus performed a phase equilibrium study on the propionic anhydride/CO2 system, at temperatures between 35 and 50 oC. This study revealed that, at 35 oC and pressures from 7 MPa, the system is monophasic for all compositions. The enzymatic catalysis studies carried out with propionic anhydride revealed that the extent of noncatalyzed reaction was high, with a negative effect on enantioselectivity. These studies showed also that it was possible to reduce considerably the impact of the noncatalyzed reaction relative to the reaction catalyzed by CRL by lowering temperature to 4 oC. Vinyl decanoate was shown to lead to the best results at conditions amenable to a process combining the use of supercritical CO2 as agent for post-reaction separation. The use of vinyl decanoate in a number of IL solvents, namely [bmim][PF6], [bmim][BF4], [hmim][PF6], [omim][PF6], and [bmim][Tf2N], led to an enantiomeric excess of product (eep) values of over 96%, at about 50% conversion, using CRL. In n-hexane and supercritical CO2, reaction progressed more slowly.(...)

Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.