Colorectal cancer (CRC) is characterised as a type of carcinoma occurring in 60 – 65% of the reported cases. CRC develops through various genetic and biochemical processes being the adenoma-carcinoma sequence the most common development pathway, having adenoma as a precursor of the CRC. In Portugal, in 2020, colon cancer ranked 2ⁿᵈ as the most diagnosed cancer followed by rectal cancer ranked 6ᵗʰ. The metabolites produced by the gut microbiome can interfere with cellular functions, cell proliferation and/or decrease inflammation, contributing to a propitious biochemical environment for CRC. In this study, the nuclear magnetic resonance (NMR) metabolomic technique, allows the study of the metabolites, including the ones existing in biological samples. NMR is highly reproducible, has a high throughput, and it's easy to automate and can quantify a great number of metabolites. Stool samples have a complex composition, including metabolites. They can provide information related to the diverse processes of the individual. Stool samples are at risk of deterioration after the collection process, being necessary a storage method that can guarantee its integrity to posterior analysis. Therefore, the first objective of this study was to determine the impact of the storage at 4ºC on the metabolomic profiles of these samples. Urine samples contain various compounds, such as metabolites from diverse biological processes. Variations in their metabolic profiles related to CRC have been already observed, suggesting that can be possibly useful in the CRC diagnosis. Therefore, the second objective of this study was to identify variations in the urine metabolomic profiles and their ability to be used in the CRC diagnosis. Six faecal samples were stored at 4ºC for 24 and 48 hours (h) to test their influence on the stability of the metabolomic profiles. The NMR analysis showed that the metabolomic integrity of the samples was maintained, suggesting that the storage at 4ºC up to 48h does not affect the metabolomic viability and integrity of the stool samples. In a second approach, forty-nine urine samples from a Portuguese cohort were analysed to identify metabolites that could be used as CRC biomarkers. Three clinical groups, healthy, adenoma, and CRC, were analysed through the NMR technique. Distinct profiles could be identified for each clinical group. The metabolites hippurate and creatinine, as well as ascorbate, presented different concentrations between the CRC and adenoma being possible biomarkers for variations in the disease progression.

Urine is a biofluid easy to collect through a non-invasive technique that allows collecting a large volume of sample. The use of urine for disease diagnosis is not yet well explored. However, it has gained attention over the last three years. It has been applied in the diagnosis of several illnesses such as kidney disease, bladder cancer, prostate cancer and cardiovascular diseases. In the last decade, gold nanoparticles (Au NPs) have attracted attention in biosensors’ development for the diagnosis of diseases due to their electrical and optical properties, ability to conjugate with biomolecules, high sensitivity, and selectivity. Therefore, this article aims to present a comprehensive view of state of the art on the advances made in the quantification of analytes in urinary samples using AuNPs based assays, with a focus on protein analysis. The type of diagnosis methods, the Au NPs synthesis approaches and the strategies for surface modification aiming at selectivity towards the different targets are highlighted.

In this present study, magnetic Fe3O4@SiO2 nanoparticles (MNPs) functionalized with octadecyl groups (Fe3O4@SiO2-C18 NPs) were synthesized, characterized and employed, for the first time, as powerful nanosorbent to extract endogenous volatile organic metabolites (EVOMs) namely, hexanal, heptanal, decanal, benzaldehyde, 4-heptanone, 5-methyl-2-furfural and phenol, described as potential biomarkers of cancer, from human urine. By using co-precipitation, surface modification methods, the carbon-ferromagnetic nanocomposite was synthesized and characterized by infrared spectrum (IR) and transmission electron microscopy (TEM). By coupling with gas chromatography-mass spectrometry (GC-qMS), a reliable, sensitive and cost-effective method was validated. To test the extraction efficiency of the carbon-ferromagnetic nanocomposite toward urinary EVOMs experimental variables affecting the extraction performance, including nanosorbent amount, adsorption time, elution time, and nature of elution solvent, were investigated in detail. The extraction process was performed by dispersing Fe3O4@SiO2-C18 NPs into working solution containing targeted VOMs, and into urine samples, and then eluted with an adequate organic solvent. The eluate was collected, concentrated and analyzed by GC-qMS. Under the optimized conditions, the LODs and LOQs achieved were in the range of 9.7-57.3 and 32.4-190.9ng/mL, respectively. Calibration curves were linear (r(2)≥0. 988) over the concentration ranges from 0.25 to 250ng/mL. In addition, a satisfying reproducibility was achieved by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 3 and 11%, respectively. The method also afforded satisfactory results in terms of the matrix effect (72.8-96.1%) and recoveries (accuracy) higher than 75.1% for most of the studied EVOMs. The Fe3O4@SiO2-C18 NPs-based sorbent extraction combined with GC-qMS revealed that the new nanosorbent had a strong ability to retain the target metabolites providing a new, reliable and high throughput strategy for isolation of targeted EVOMs in human urine, suggesting their potential to be applied in other EVOMs.

A sensitive assay to identify volatile organic metabolites (VOMs) as biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. Therefore the aim of this study was to establish the urinary metabolomic profile of breast cancer patients and healthy individuals (control group) and to explore the VOMs as potential biomarkers in breast cancer diagnosis at early stage. Solid-phase microextraction (SPME) using CAR/PDMS sorbent combined with gas chromatography–mass spectrometry was applied to obtain metabolomic information patterns of 26 breast cancer patients and 21 healthy individuals (controls). A total of seventy-nine VOMs, belonging to distinct chemical classes, were detected and identified in control and breast cancer groups. Ketones and sulfur compounds were the chemical classes with highest contribution for both groups. Results showed that excretion values of 6 VOMs among the total of 79 detected were found to be statistically different (p < 0.05). A significant increase in the peak area of (−)-4-carene, 3-heptanone, 1,2,4-trimethylbenzene, 2-methoxythiophene and phenol, in VOMs of cancer patients relatively to controls was observed. Statiscally significant lower abundances of dimethyl disulfide were found in cancer patients. Bioanalytical data were submitted to multivariate statistics [principal component analysis (PCA)], in order to visualize clusters of cases and to detect the VOMs that are able to differentiate cancer patients from healthy individuals. Very good discrimination within breast cancer and control groups was achieved. Nevertheless, a deep study using a larger number of patients must be carried out to confirm the results.

In this work, the design of a microfluidic paper-based analytical device (µPAD) for the quantification of nitrate in urine samples was described. Nitrate monitoring is highly relevant due to its association to some diseases and health conditions. The nitrate determination was achieved by combining the selectivity of the nitrate reductase enzymatic reaction with the colorimetric detection of nitrite by the well-known Griess reagent. For the optimization of the nitrate determination µPAD, several variables associated with the design and construction of the device were studied. Furthermore, the interference of the urine matrix was evaluated, and stability studies were performed, under different conditions. The developed µPAD enabled us to obtain a limit of detection of 0.04 mM, a limit of quantification of 0.14 mM and a dynamic concentration range of 0.14–1.0 mM. The designed µPAD proved to be stable for 24 h when stored at room temperature in air or vacuum atmosphere, and 60 days when stored in vacuum at −20◦C. The accuracy of the nitrate µPAD measurements was confirmed by analyzing four certified samples (prepared in synthetic urine) and performing recovery studies using urine samples.

In this work, a new microfluidic paper-based analytical device (μPAD) was developed for on-hand creatinine quantification in urine samples. When compared to other conventional methods, this innovative paper device is more accessible, portable, providing low-cost analysis and applicable to non-invasive biological fluids. Furthermore, this paper approach is environmentally friendly, as it uses small amounts of reagents, results in low waste production and it is disposable by incineration. Its configuration consisted in two layers of filter paper discs of 9.5 mm of diameter, in which the top layer served as a reservoir and the bottom layer contained the reagent (alkaline picrate). This hydrophilic unit consisting of two-layer of filter paper was aligned within a laminated plastic pouch to set the hydrophobic separation, with 3 mm holes for the sample insertion. When inserted, creatinine of the standard/sample reacts with alkaline picric acid (colorimetric reaction), and the colour changes from yellow to orange/red. Under optimal conditions, the developed method allowed creatinine quantification in the dynamic range of 2.20-35.0 mg/dL, with a limit of detection (LOD) of 0.66 mg/dL and a limit of quantification (LOQ) of 2.20 mg/dL. The image was scanned after 20 minutes and was processed in ImageJ software to obtain the colour intensity developed, later converted to pseudo-absorbance. The stability of the reaction product after the sample insertion was verified until 3 hours and the device could be scanned within that time. The μPAD allows to obtain linear calibration curves up to one week when stored in vacuum and placed in the fridge at approximately 4 ºC. Finally, the method was validated by the results obtained with the developed device and a comparison method, and it was demonstrated that there were no statistically significant differences between them. As so, it was possible to conclude that this novel device was successfully developed, as it proved to be sensitive and simple, as well as affordable, as it showed a cost of approximately 0.40 € (in terms of consumables).

In this work, a potentiometric flow injection method is described for the fast bi-parametric determination of iodide and iodate in urine and salt samples. The developed methodology aimed for iodine speciation with a potentially portable system (running on batteries). The iodate reduction to iodide was effectively attained in line within the same manifold. The iodide determination was accomplished in the dynamic range of 2.50×10−6– 1.00×10−3 M and the total iodine dynamic range, resulted from iodide plus iodate, was 3.50×10−6– 2.00×10−3 M. The calculated limits of detection were 1.39×10−6 M and 1.77×10−6 M for iodide and iodate, respectively. A determination rate of 21 h−1 for the bi-parametric iodide and iodate determination was obtained for sample injection. The urine samples (RSD < 5.8% for iodide and RSD < 7.0% for iodate) results were in agreement with those obtained by the classic Sandell-Kolthoff reaction colorimetric reference procedure (RD < 7.0%) and standard samples from Centers for Disease Control and Prevention, Atlanta, USA (CDC) international inter-laboratory EQUIP program. The developed flow method was also successfully applied to the iodide and iodate determination in salt samples (RSD < 3.1% for iodate and iodide), with comparable results to conventional procedures. No significant interferences were observed interference percentage < 9% for both determinations.

In this work, a new microfluidic paper-based analytical device (µPAD) was developed for on-hand creatinine quantification in urine samples. When compared to conventional methods, this innovative paper device is more accessible and portable, it provides low-cost analysis (cost of consumables of 40 cents), and it is applicable to non-invasive biological fluids. Furthermore, the paper-based approach is used within an environmentally friendly assembly with no need for wax printing and small amounts of reagents resulting in low waste production and easy disposal by incineration. Its assembly method includes cutting paper discs arranged into several reading units within a plastic pouch, enabling effective creatinine quantification with accuracy based on a vertical flow approach. The method is based on the colourimetric reaction between creatinine and alkaline picric acid, where the solution colour changes from yellow to orange/red. Under optimal conditions, the developed method allowed creatinine quantification in the dynamic range of 2.20–35.0 mg/dL, with a limit of detection (LOD) of 0.66 mg/dL and a limit of quantification (LOQ) of 2.20 mg/dL. The colour intensity developed was processed in ImageJ software, based on digital image scanning, performed in 20 min (up to 4 h) after the sample insertion. The device is stable for up to one week when stored in a vacuum at 4 °C. The method was validated by comparing the results with a batch-wise procedure, where there were no statistically significant differences between both methods.