Design of paper-based analytical devices for chemical and biochemical assays of biomarkers in biological fluids of non-invasive collection

Ensuring basic healthcare access around the world remains a challenge, particularly in lowincome regions. In response, international initiatives such as the Sustainable Development Goals (SDGs) and the World Health Organization (WHO) guidelines emphasize the need to develop innovative, accessible, and cost-effective diagnostic tools. Point-of-care testing has emerged as an ideal solution, enabling rapid and decentralized analysis. Among these type of devices, microfluidic paper-based analytical devices (µPADs) have gained attention due to their affordability, portability, and ease of use, making them a particularly valuable tool in resource-limited locations. The purpose of the work presented throughout this thesis was to design and develop innovative point-of-care methodologies based on the µPAD approach for the determination of several healthrelated parameters, that could serve as an adding tool in the diagnosis and monitoring of several health conditions. The use of biological samples of non-invasive collection, such as saliva and urine, enhances diagnostic accessibility, particularly in point-of-care settings where traditional sample collection may be impractical or even impossible. Additionally, this thesis also explores the use of colorimetric detection methods, a straightforward approach to quantifying the targeted analytes by producing visible colour changes. To further improve the specificity and accuracy of these diagnostic tools, enzymatic reactions were incorporated into some of the developed µPADs. The first developed device was dedicated to the quantification of total iron in urine samples by using the colorimetric reaction of bathophenanthroline with iron (II) coupled with hydroxylamine, a well-known reducing agent capable of converting iron (III) in iron (II). To handle the potential colour of the urine samples, a sample blank was included in the device. This feature was vital to ensure the applicability of the developed μPAD, as urine may present a wide variability of colour range, from light yellow to brownish. The determination of nitrate was also accomplished in urine samples with a newly developed µPAD. This device included the enzymatic reaction of nitrate reductase to perform the conversion of nitrate to nitrite and the Griess reagent which provided the colorimetric detection of the resulting nitrite. In order to delay the vertical flow and increase the extent of the enzymatic reaction, a hydrophilic membrane layer was also incorporated into the device. The small porosity of this membrane also led to the retention of the compound responsible for the colour of urine, enabling a direct analysis of the samples. The next devices developed performed the quantification of NHX and urea in saliva samples but with a more complex approach. Not only were the devices composed of four layers, but the detection relies on the diffusion of NH3 (g) through a gas-diffusion membrane to produce a colorimetric change of a pH indicator. The hydrophobicity of this membrane also helped eliminate possible interferences of the saliva sample in the colorimetric reaction, since it did not allow the sample to pass through the membrane and reach the colour reagent. The urea determination was accomplished by including urease in the device, since it selectively catalyzes the dissociation of urea in ammonia and carbon dioxide. The urease activity determination was accomplished using a similar structure and reactions to the one used for the urea µPAD. However, the determination itself was achieved using the kinetic capabilities of the enzyme by correlating to the urease activity with the variation of the signal obtained between two different enzymatic reaction times. This strategy not only allowed a more accurate quantification but also suppressed the influence of NHX already present in the samples. The last device developed using the µPAD structure approach was to quantify glucose in saliva samples. With only two layers in its composition, this device uses a combination of two enzymatic reactions. First, glucose oxidase converts glucose into gluconic acid and hydrogen peroxide, then followed by the release of oxygen from hydrogen peroxide performed by peroxidase. The colorimetric detection is accomplished with the oxidation of o-dianisidine. In this work, a correlation between the concentration of glucose in saliva and blood was also successfully established, using #5 saliva samples of diabetic patients and the correspondent glucometer measurements provided by the patients. A rather different approach was used to perform the semi-quantitative analysis of E. coli DNA. Instead of developing a µPAD, an option was made to design a barcode-style lateral flow strip that would allow the semi-quantitative detection of E. coli. The amplification of the DNA in the sample was performed using the Loop-Mediated Isothermal Amplification (LAMP) technique since it can be performed at a constant temperature and provides high sensitivity and specificity results. The developed methodology, although requiring further optimization, showed reliable performance and offers a rapid, cost-effective method for E. coli detection. Overall, this thesis demonstrates the potential of microfluidic paper-based analytical devices and lateral flow assays as innovative, cost-effective diagnostic tools for point-of-care applications. By addressing critical challenges such as sample complexity, reagent stability, and measurement accuracy, the developed devices enhance accessibility to reliable testing, particularly in resourcelimited settings. Their affordability and ease of use further emphasize their role in expanding diagnostic capabilities outside traditional laboratory environment.