Microfluidic Paper-Based Device Incorporated with Silica Nanoparticles for Iodide Quantification in Marine Source Dietary Supplements.

Iodine is an essential micronutrient for humans due to its fundamental role in the biosynthesis of thyroid hormones. As a key parameter to assess health conditions, iodine intake needs to be monitored to ascertain and prevent iodine deficiency. Iodine is available from various food sources (such as seaweed, fish, and seafood, among others) and dietary supplements (multivitamins or mineral supplements). In this work, a microfluidic paper-based analytical device (µPAD) to quantify iodide in seaweed and dietary supplements is described. The developed µPAD is a small microfluidic device that emerges as quite relevant in terms of its analytical capacity. The quantification of iodide is based on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide in the presence of iodine, which acts as the catalyst to produce the blue form of TMB. Additionally, powder silica was used to intensify and uniformize the colour of the obtained product. Following optimization, the developed µPAD enabled iodide quantification within the range of 10-100 µM, with a detection limit of 3 µM, and was successfully applied to seaweeds and dietary supplements. The device represents a valuable tool for point-of-care analysis, can be used by untrained personnel at home, and is easily disposable, low-cost, and user-friendly.

Combining microfluidic paper-based platform and metal-organic frameworks in a single device for phenolic content assessment in fruits.

A microfluidic paper-based device (µPAD) has been combined with metal-organic frameworks (MOFs) for total phenolic compounds (TPC) quantification in fruit samples for the first time. The performance of the µPAD, based upon the vertical flow approach, was enhanced in order to determine the TPC content with high accuracy in fruit samples. The method was based on the traditional Folin-Ciocalteu Index using gallic acid or oenotannin as reference phenolic compounds. This novel design and construction of the device are in agreement with the principles of Green Chemistry avoiding wax technology (lower toxicity). The analytical parameters that affect the colorimetric method (using digital imaging of the colored zone) performance were optimized including design, sample volume, and MOF amount. Then, the analytical features of the developed method were investigated such as dynamic range (1.6-30 mg L-1), limit of detection (0.5 mg L-1), and precision (RSD < 9%). Besides, the in-field analysis is achievable with a color stability up to 6 h after the loading process of the sample and storage stability for at least 15 days without performance losses (under vacuum at - 20 °C). Furthermore, the MOF ZIF-8@paper was characterized to study its composition and the successful combination. The feasibility of the proposed method was demonstrated by determining the TPC in 5 fruit samples using oenotannin as reference solute. The accuracy was validated by comparison of the data with the results obtained with the recommended protocol proposed by the International Organisation of Vine and Wine (OIV).

Chip-Based Spectrofluorimetric Determination of Iodine in a Multi-Syringe Flow Platform with and without In-Line Digestion-Application to Salt, Pharmaceuticals, and Algae Samples.

In this work, a flow-based spectrofluorimetric method for iodine determination was developed. The system consisted of a miniaturized chip-based flow manifold for solutions handling and with integrated spectrofluorimetric detection. A multi-syringe module was used as a liquid driver. Iodide was quantified from its catalytic effect on the redox reaction between Ce(IV) and As(III), based on the Sandell-Kolthoff reaction. The method was applied for the determination of iodine in salt, pharmaceuticals, supplement pills, and seaweed samples without off-line pre-treatment. An in-line oxidation process, aided by UV radiation, was implemented to analyse some samples (supplement pills and seaweed samples) to eliminate interferences and release iodine from organo-iodine compounds. This feature, combined with the fluorometric reaction, makes this method simpler, faster, and more sensitive than the classic approach of the Sandell-Kolthoff reaction. The method allowed iodine to be determined within a range of 0.20-4.0 µmol L-1, with or without the in-line UV digestion, with a limit of detection of 0.028 µmol L-1 and 0.025 µmol L-1, respectively.

A Robust Flow-Based System for the Spectrophotometric Determination of Cr(VI) in Recreational Waters.

A flow-based method for the spectrophotometric determination of chromium (VI) in recreational waters with different salinities was developed. Chromium can occur in the environment in different oxidation states with different related physiological properties. With regard to chromium, the speciation is particularly important, as the hexavalent chromium is considered to be carcinogenic. To achieve that purpose, the use of the diphenylcarbazide (DPC) selective colored reaction with the hexavalent chromium was the chosen strategy. The main objective was to develop a direct and simple spectrophotometric method that could cope with the analysis of different types of environmental waters, within different salinity ranges (fresh to marine waters). The potential interference of metal ions, that can usually be present in environmental waters, was assessed and no significant interferences were observed (<10%). For a complete Cr(VI) determination (three replicas) cycle, the corresponding reagents consumption was 75 µg of DPC, 9 mg of ethanol and 54 mg of sulfuric acid. Each cycle takes about 5 min, including the system clean-up. The limit of detection was 6.9 and 12.2 µg L−1 for waters with low and high salt content, respectively. The method was applied for the quantification of chromium (VI) in both fresh and marine water, and the results were in agreement with the reference procedure.

New microfluidic paper-based analytical device for iron determination in urine samples.

Iron is an important micronutrient involved in several mechanisms in the human body and can be an important biomarker. In this work, a simple and disposable microfluidic paper-based analytical device (µPAD) was developed for the quantification of iron in urine samples. The detection was based on the colorimetric reaction between iron(II) and bathophenanthroline and the reduction of iron(III) to iron(II) with hydroxylamine. The developed µPAD enabled iron determination in the range 0.07-1.2 mg/L, with a limit of detection of 20 µg/L and a limit of quantification of 65 µg/L, thus suitable for the expected values in human urine. Additionally, targeting urine samples, the potential interference of the samples color was overcome by incorporating a sample blank assessment for absorbance subtraction. Stability studies revealed that the device was stable for 15 days prior to usage and that the formed colored product was stable for scanning up to 3 h. The accuracy of the developed device was established by analyzing urine samples (#26) with the developed µPAD and with the atomic absorption spectrometry method; the relative deviation between the two sets of results was below 9.5%.

Design and Functionalization of a µPAD for the Enzymatic Determination of Nitrate in Urine.

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.

Simultaneous nitrification and phosphate removal by bioaugmented aerobic granules treating a fluoroorganic compound.

The presence of toxic compounds in wastewater can cause problems for organic matter and nutrient removal. In this study, the long-term effect of a model xenobiotic, 2-fluorophenol (2-FP), on ammonia-oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and phosphate accumulating organisms (PAO) in aerobic granular sludge was investigated. Phosphate (P) and ammonium (N) removal efficiencies were high (>93%) and, after bioaugmentation with 2-FP degrading strain FP1, 2-FP was completely degraded. Neither N nor P removal were affected by 50 mg L-1 of 2-FP in the feed stream. Changes in the aerobic granule bacterial communities were followed. Numerical analysis of the denaturing gradient gel electrophoresis (DGGE) profiles showed low diversity for the ammonia monooxygenase (amoA) gene with an even distribution of species. PAOs, including denitrifying PAO (dPAO), and AOB were present in the 2-FP degrading granules, although dPAO population decreased throughout the 444 days reactor operation. The results demonstrated that the aerobic granules bioaugmented with FP1 strain successfully removed N, P and 2-FP simultaneously.

Micro-PAD card for measuring total ammonia nitrogen in saliva.

This work presents a portable microfluidic paper-based analytical device (micro-PAD) card for the quantification of total ammonia nitrogen in human saliva. The amount of total ammonia nitrogen in saliva can be an indicator of the status of the oral microbiome with potential correlation to kidney health problems. The developed micro-PAD card comprises twenty units consisting of three stacked layers of circular discs: the sample layer, paper discs impregnated with sodium hydroxide solution, the PTFE membrane layer, and the detection layer, paper discs impregnated with bromothymol blue. The twenty units were aligned on transparent laminating pouches laminated to form the micro-PAD card (7.5 cm × 10.5 cm). Saliva samples can be directly dispensed onto the micro-PAD card and the detection was achieved by the BTB indicator color change, from yellow to blue, after conversion of ammonium into ammonia and diffusion of the ammonia gas through a hydrophobic layer. The determination of total ammonia nitrogen in saliva using the developed micro-PAD card intended to be very simple method and operated without the need of laboratory equipment. A quantification limit of 11.3 NH4+mg L-1 and linear application range from up to 150 NH4+mg L-1 were obtained making it suitable for the expected concentrations of total ammonia nitrogen in human saliva. It was successfully applied to saliva samples and its validation obtained by comparison against a potentiometric method. The card is stable for at least 1 month making it ideal as a portable device for point-of-care diagnosis. Graphical Abstract.

Use of a Polymer Inclusion Membrane and a Chelating Resin for the Flow-Based Sequential Determination of Copper(II) and Zinc(II) in Natural Waters and Soil Leachates.

A bi-parametric sequential injection method for the determination of copper(II) and zinc(II) when present together in aqueous samples was developed. This was achieved by using a non-specific colorimetric reagent (4-(2-pyridylazo)resorcinol, PAR) together with two ion-exchange polymeric materials to discriminate between the two metal ions. A polymer inclusion membrane (PIM) and a chelating resin (Chelex 100) were the chosen materials to retain zinc(II) and copper(II), respectively. The influence of the flow system parameters, such as composition of the reagent solutions, flow rates and standard/sample volume, on the method sensitivity were studied. The interference of several common metal ions was assessed, and no significant interferences were observed (<10% signal deviation). The limits of detection were 3.1 and 5.6 µg L-1 for copper(II) and zinc(II), respectively; the dynamic working range was from 10 to 40 µg L-1 for both analytes. The newly developed sequential injection analysis (SIA) system was applied to natural waters and soil leachates, and the results were in agreement with those obtained with the reference procedure.

A total analytical system featuring a novel solid-liquid extraction chamber for solid sample flow analysis.

In this work, a total flow analysis system based on a novel solid-liquid extraction chamber is presented. This strategy enables all the main experimental procedures for the analysis of a solid sample to be performed automatically: enrichment of the liquid extract, sample treatment, filtration of the liquid extract from the solid sample, directing the extract towards detection, and finally cleansing of the chamber for the following solid sample to be analyzed. The chamber designed to be incorporated in the flow manifold presents two main features: it accommodates stirring bars for enhancing the extraction process, and it presents replaceable solid sample containers (a spare part of the solid-liquid extraction chamber) to easily replace the solid sample and therefore enhance sample analysis throughput. The chamber performance was assessed using two different solid samples, an ion exchanger resin and vegetable samples, focussing on proton and nitrate ion extraction, respectively. The main figures of merit achieved were relative standard deviation (RSD) and relative error values below 7 % for all determinations. The determination rate for vegetable samples was ca. 12 samples h-1. The proposed strategy may be exploited to perform automatically the analysis of solid samples as it embodies a simple automatic strategy of a very important but time-consuming and laborious analytical operation. Graphical abstract TAS for solid liquid extraction and nitrate potentiometric determination of vegetable samples.

Use of a rhodamine-based chelator in a microfluidic paper-based analytical device for the in-situ copper quantification in natural waters.

This work describes the development of a microfluidic paper-based analytical device (µPAD) for the determination of copper in fresh and marine waters. A functionalized rhodamine-based chelator was synthesized and used as a chromogenic reagent, forming a highly intense pink complex with the analyte. The aim was to create a paper device that offers optimal performance and provides in-situ, rapid and cost-effective analysis in line with World Health Organization guidelines. The influence on the determination of several physical and chemical parameters was evaluated aiming to achieve the best performance. Under optimised conditions, a linear correlation was established in the range of 0.05-0.50 mg L-1 of copper, with a limit of detection of 10 µg L-1. The accuracy of the proposed method was assessed by comparing the results obtained with the developed µPAD and the results obtained with Inductively Coupled Plasma measurements (RE < 10 %). Recovery studies were also performed using different types of water samples with no need for any prior sample pre-treatment: tap, well, river and seawater. The average recovery percentage of 101 % (RSD = 4.3 %) was obtained, a clear indication of no multiplicative matrix interferences.

Performance of an aerobic granular sequencing batch reactor fed with wastewaters contaminated with Zn2+.

The main aim of this study was to investigate the performance of an aerobic granular sludge sequencing batch reactor (AGS-SBR) receiving water streams supplied with different loads of Zn(2+) (50 and 100 mg L(-1)) during an operation of 866 cycles (ca. 109 days). When the metal was not fed, chemical oxygen demand (COD), PO4(3-) and NH4(+) were efficiently removed, with efficiencies of 56, 23 and 72% respectively. DGGE profiles showed that Zn(2+) supply negatively affected the bacterial diversity and community structure of the granules. Consequently, the shock loadings with Zn(2+), particularly at the higher levels (100 mg L(-1)), affected the nutrient removal in the AGS-SBR, although the reactor still generally complied with admissible legal values concerning organic matter, nitrogen and Zn. Simultaneous removal of PO4(3-) and TSS in such conditions needs further refining but the application of aerobic granular SBR in the treatment of Zn(2+) contaminated wastewaters seems viable.

Non-transferrin-bound iron determination in blood serum using microsequential injection solid phase spectrometry- proof of concept.

Non-transferrin-bound iron (NTBI) is a group of circulating toxic iron forms, which occur in iron overload or health conditions with dysregulation of iron metabolism. NTBI is responsible for increased oxidative stress and tissue iron loading. Despite its relevance as a biochemical marker in several diseases, a standardized assay is still lacking. Several methods were developed to quantify NTBI, but results show high inter-method and even inter-laboratory variability. Thus, the development of a consistent NTBI assay is a major goal in the management of iron overload and related clinical conditions. In this work, a micro sequential injection lab-on-valve (µSI-LOV) method in a solid phase spectrophotometry (SPS) mode was developed for the quantification of NTBI, using a bidentate 3,4-hydroxypyridinone (3,4-HPO) ligand anchored to sepharose beads as a chromogenic reagent. To attain SPS, the functionalized beads were packed into a column in the flow cell, and the analyte, NTBI retained as iron (III), formed a colored complex at the beads while eliminating the sample matrix. The dynamic concentration range was 1.62-7.16 µmol L-1 of iron (III), with a limit of detection of 0.49 µmol L-1 and a limit of quantification of 1.62 µmol L-1. The proposed µSI-LOV-SPS method is a contribution to the development of an automatic method for the quantification of the NTBI in serum samples.

Assessment of collagen content in fish skin - development of a flow analysis method for hydroxyproline determination.

This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L-1, with a limit of detection (LOD) of 2.6 mg L-1 and a limit of quantification (LOQ) of 23.8 mg L-1. Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).

Sequential injection spectrophotometric method for screening bromate produced as an ozonation water disinfection by-product.

The aim of this work was the development of an automatic sequential injection analysis method to monitor the ozonation process for water disinfection. The determination was based on the reaction between bromate and o-dianisidine in the presence of bromide in acidic medium. The determination parameters were studied and adjusted to enable bromate quantification in the range 0.35-4.0 mg BrO3-/L with a limit of detection of 20 µg BrO3-/L. The choice of a sequential injection procedure enabled a minimal consumption of reagents and no need for sample pre-treatment. The developed sequential injection proved to be accurate with < 5% relative deviation when compared to ICP-MS and an average of 101% in recovery percentages studies. It was effectively applied to monitor an ozonation process enabling the follow-up of the process with real-time quantification of the bromate content.

Sequential injection method for bi-parametric determination of iron and manganese in soil leachates.

The aim of this work was to develop a sequential injection (SI) method for the determination of the micronutrients iron and manganese, in soil leachates, as a tool to assess potential groundwater contamination. The described sequential injection method was based on the reaction of iron with chelator MRB12, a greener alternative chromogenic reagent, and the reaction of manganese with zincon, within a single manifold. The developed SI method enabled the determination of iron in the range 0.10-1.00 mg L-1, and manganese in the range 0.25-2.5 mg L-1 with a limit of detection of 0.08 mg L-1 for iron and 0.24 mg L-1 for manganese. The determination of both parameters was made in 6 minutes, in triplicate. The application to monitor laboratory scale soil core columns (LSSCs), as a simulation of the soil leaching process, proved its efficiency to assess potential contamination of ground waters. Iron and manganese contents were effectively analysed in two different scenarios to mimic the leaching process with rainwater and fertilizer.

Design of a microfluidic paper-based device for the quantification of phenolic compounds in wine samples.

In this work, the design and development of a microfluidic paper-based device (µPAD) for the quantification of total phenolic compounds (TPC) in wines is described. The developed µPAD was based upon the vertical flow concept and the colour reaction used was the known Folin-Ciocalteu reaction using gallic acid as reference phenolic compound. After studying operational parameters, namely type of paper, reagents and sample volume, a dynamic range of 5-50 mg L-1 was obtained with a limit of detection of 1.2 mg L-1. The described device proved to have good precision (relative standard deviation < 5%) and no significant interferences were observed from known compounds present in wines. Furthermore, the stability of the colour product and of the device itself were assessed; the µPAD was stable for 30 days (in the dark at room temperature) and it could be scanned up to 8 h after sample introduction. The developed µPAD pose as a simple method for TPC quantification and was successfully applied to several wine samples including sparkling and table wines with two different approaches: i) using gallic acid as reference compound with standard addition; and ii) using taniraisin with external calibration. The accuracy of the proposed µPAD method was assessed by comparison with the reference spectrophotometric method according to the International Organisation of Vine and Wine (OIV) recommendations.

Characterization of Oral Enterobacteriaceae Prevalence and Resistance Profile in Chronic Kidney Disease Patients Undergoing Peritoneal Dialysis.

Chronic Kidney Disease (CKD) is a growing public-health concern worldwide. Patients exhibit compromised immunity and are more prone to infection than other populations. Therefore, oral colonization by clinically relevant members of the Enterobacteriaceae family, major agents of both nosocomial and dialysis-associated infections with frequent prevalence of antibiotic resistances, may constitute a serious risk. Thus, this study aimed to assess the occurrence of clinically relevant enterobacteria and their antibiotic resistance profiles in the oral cavity of CKD patients undergoing peritoneal dialysis (CKD-PD) and compare it to healthy controls. Saliva samples from all the participants were cultured on MacConkey Agar and evaluated regarding the levels of urea, ammonia, and pH. Bacterial isolates were identified and characterized for antibiotic resistance phenotype and genotype. The results showed that CKD-PD patients exhibited significantly higher salivary pH, urea, and ammonia levels than controls, that was accompanied by higher prevalence and diversity of oral enterobacteria. Out of all the species isolated, only the prevalence of Raoultella ornithinolytica varied significantly between groups, colonizing the oral cavity of approximately 30% of CKD-PD patients while absent from controls. Antibiotic resistance phenotyping revealed mostly putative intrinsic resistance phenotypes (to amoxicillin, ticarcillin, and cephalothin), and resistance to sulfamethoxazole (~43% of isolates) and streptomycin (~17%). However, all isolates were resistant to at least one of the antibiotics tested and multidrug resistance isolates were only found in CKD-PD group (31,6%). Mobile genetic elements and resistance genes were detected in isolates of the species Raoultella ornithinolytica, Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and Enterobacter asburiae, mostly originated from CKD-PD patients. PD-related infection history revealed that Enterobacteriaceae were responsible for ~8% of peritonitis and ~ 16% of exit-site infections episodes in CKD-PD patients, although no association was found to oral enterobacteria colonization at the time of sampling. The results suggest that the CKD-induced alterations of the oral milieu might promote a dysbiosis of the commensal oral microbiome, namely the proliferation of clinically relevant Enterobacteriaceae potentially harboring acquired antibiotic resistance genes. This study highlights the importance of the oral cavity as a reservoir for pathobionts and antibiotic resistances in CKD patients undergoing peritoneal dialysis.

Novel microfluidic paper-based analytical devices (µPADs) for the determination of nitrate and nitrite in human saliva.

In this work, two different microfluidic paper-based analytical devices (µPADs) were developed for the quantification of nitrite and nitrate in human saliva samples, in order to aid in the diagnosis of some diseases and health conditions associated with these ions. The development of these nitrite and nitrate µPADs involved several studies to optimize their design and construction, including an interference assessment and stability studies. These µPADs allowed a nitrite determination in a range of 5-250 µM with limits of detection and quantification of 0.05 µM and 0.17 µM, respectively, and a nitrate determination in the range 0.2-1.2 mM with limits of detection and quantification of 0.08 mM and 0.27 mM, respectively. As for the stability, both of the µPADs were stable when stored in vacuum at 4 °C (the nitrite µPAD for at least 60 days and the nitrate µPAD for at least of 14 days) and, after the sample placement, the nitrite and nitrate µPADs could be scanned within the first 4 and 2 h, respectively. The nitrite µPAD measurements were compared with the ones obtained from the standard colorimetric method and there were no statistically significant differences between these two methods. To evaluate the accuracy of nitrate µPAD measurements, 4 certified water samples were used and recovery studies using saliva samples were performed.

Greener and wide applicability range flow-based spectrophotometric method for iron determination in fresh and marine water.

A flow-based method for the spectrophotometric determination of iron in recreational waters, both fresh and marine (variable salinity content), was developed. For that purpose, 3-hydroxy-4-pyrydinone ligand functionalized with an ether function was synthetized and used as chromogenic chelator (1-(3'-methoxypropyl)-2-methyl-3-benzyloxy-4-(1H)pyridinone - MRB13) for iron quantification. This water-soluble reagent was previously reported as a greener alternative to quantify iron, due to its low toxicity and a more environmental friendly synthesis. Furthermore, it also displayed a high affinity and specificity for iron. With the main objective of quantifying iron in a variety of water types (different matrices and iron content), two strategies were developed, one of them including on-line solid-phase extraction (SPE), and the other without resorting to a SPE process. Water matrix clean-up and iron enrichment was achieved using a nitrilotriacetic acid resin column. The potential interference of metal ions usually present in water samples was assessed and no significant interference (<10%) was observed. The limits of detection were 11 and 2.9 µg L-1 without and with SPE, respectively. For one determination (three replicates), the corresponding consumption of MRB13 is 90 µg, sodium hydroxide is 1.4 mg, and boric acid is 5.6 mg. The method was applied to certified water samples and the results were in agreement with certified values. The developed method was also applied to fresh and marine water, and recovery ratios of 103 ± 4 and 101 ± 7 without and with SPE, respectively, were achieved.