M. Lúcia M.F.S. Saraiva

Enzyme based assays in a sequential injection format: a review.

Sequential injection analysis systems have been extensively exploited in the last decades for the implementation of enzyme based assays aiming the evaluation of enzyme activity or the determination of specific analytes. The most prominent aspects of the automation of enzymatic assays in these systems are discussed in this review. Special attention is devoted to the mode of enzyme manipulation in homogeneous or heterogeneous media and to the comparison with batch and flow injection enzyme methodologies. The possibility of implementing strategies for the enhancement of selectivity in specific determinations is also addressed. The more recent trends in this field are discussed focusing mainly on the miniaturization resorting to the lab-on valve platform as well as on the bead injection concept.

Determination of total and oxidized glutathione in human whole blood with a sequential injection analysis system

This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between the tGSH and GSSG concentrations. The determination was based on the DTNB-GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (lambda=412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions. The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 microM, with detection limits of 0.031 and 0.014 microM, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.)<5.0% (n=10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure.

Automated high-throughput Vibrio fischeri assay for (eco)toxicity screening: application to ionic liquids.

An automated high-throughput Vibrio fischeri assay was developed and further applied to the evaluation of ionic liquids (ILs) (eco)toxicity. The assay was based on the reduction of bacterial bioluminescence in the presence of test compounds and the results were presented as EC(50). The assays were performed with eight commercially available ILs with distinct cationic head groups, alkyl side chains and anions. EC(50) values between 6.5 and 691.9 mmol L(-1) were obtained for the tested ILs, being hmim [Cl] the most toxic and bmim [Cl] the less toxic ones, confirming the influence of the different structural elements. Moreover, all the tested ILs exhibited a (eco)toxicity lower than Cu(II), used as a positive control during the optimization and analysis steps. The automated assay assured the precise control of the contact time between V. fischeri and test compound by means of a simple protocol that guaranteed adequate aspiration and handling of the solutions as well as the precise implementation of a computer controlled stop period. Furthermore, a significant reduction of the assay costs was achieved through automation mainly by a drastic reduction of the volume of bacterial suspension and test compound. The methodology was validated by comparison with a microplate assay; it was stated that the results, obtained after a 3min contact time, changed proportionally relatively to Cu(II) in both assays. This confirmed the applicability of the methodology as an (eco)toxicity screening assay, with reduction of time and increase of robustness and repeatability (n=10; rsd<1.1%). It is expected that due to its simplicity and reduced cost the developed assay can be integrated in the early stage of development of new compounds as a rapid screening test.

Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully automated methodology

A fully automated Vibrio fischeri methodology based on sequential injection analysis (SIA) has been developed. The methodology was based on the aspiration of 75 μL of bacteria and 50 μL of inhibitor followed by measurement of the luminescence of bacteria. The assays were conducted for contact times of 5, 15, and 30 min, by means of three mixing chambers that ensured adequate mixing conditions. The optimized methodology provided a precise control of the reaction conditions which is an asset for the analysis of a large number of samples. The developed methodology was applied to the evaluation of the impact of a set of ionic liquids (ILs) on V. fischeri and the results were compared with those provided by a conventional assay kit (Biotox(®)). The collected data evidenced the influence of different cation head groups and anion moieties on the toxicity of ILs. Generally, aromatic cations and fluorine-containing anions displayed higher impact on V. fischeri, evidenced by lower EC50. The proposed methodology was validated through statistical analysis which demonstrated a strong positive correlation (P>0.98) between assays. It is expected that the automated methodology can be tested for more classes of compounds and used as alternative to microplate based V. fischeri assay kits.

Estimation of postmortem interval by hypoxanthine and potassium evaluation in vitreous humor with a sequential injection system.

The estimation of the time since death known as postmortem interval (PMI) is a main issue in the field of forensic science and legal medicine. In this work it is proposed a sequential injection system for the determination of hypoxanthine and potassium in the same sample of vitreous humor since the concentrations of both parameters change with PMI and the vitreous humor has been regarded as the ideal extracellular fluid for these kinds of determinations. By measuring both parameters the accuracy of estimation of PMI can be increased, and the effects of factors which influence the values in postmortem chemistry minimized. Hypoxanthine determination is based on its oxidation to uric acid (290 nm), catalyzed by immobilized xanthine oxidase, and the quantification of potassium levels in vitreous humor was performed using a tubular potassium ion-selective electrode. With a unique analytical cycle both analytes were evaluated being potassium levels determined during the degradation of hypoxanthine in the enzymatic reactor. Working concentration ranges between 6.04-40.00 micromol L(-1) and 7.00 x 10(-5) to 1.00 x 10(-1)mmol L(-1) were obtained, for hypoxanthine and potassium, respectively. The method proved to be reproducible with R.S.D. <5% for hypoxanthine and <3% for potassium. Sampling rate was approximately 30 per hour for the sequential determination of both parameters being 15 and 60 determinations per hour if hypoxanthine or potassium, where evaluated independently. Statistical evaluation at the 95% confidence level showed good agreement between the results obtained, for the vitreous humor samples, with both the SIA system and the comparison batch procedures. Moreover the methodology has low environmental impact in agreement with the demands of green analytical chemistry as only 2.7 mL of chemical waste is produced during both determinations.

Active pharmaceutical ingredients based on salicylate ionic liquids: insights into the evaluation of pharmaceutical profiles

This work is based on the evaluation of the protein binding affinity, partition coefficients (with a biomimetic membrane) and surfactant properties of three pharmaceutically active ionic liquids based on the salicylate anion. Fluorescence spectroscopy was used for the evaluation of the binding of ionic liquids to human serum albumin and for the determination of critical micelle concentrations. Partition coefficients were determined using micelles of hexadecylphosphocholine and UV-Vis derivative spectroscopy. The results indicate that all the compounds bind strongly and spontaneously to human serum albumin and exhibit the ability to form micelles. The determined partition coefficients were up to 6 times higher than those of the starting materials, evidencing that the ionic liquid form has greater affinity for the lipid phase than the inorganic salt form of salicylate. Generally, the studied salicylate ionic liquids exhibit an interesting pharmaceutical profile presenting favorable properties regarding the incorporation of the compounds in antimicrobial pharmaceutical formulations. It was evidenced that the tested ionic liquids can exert direct effects on cell membranes as indicated by their surfactant properties and high ability to partition to hydrophobic environments.

Environmental Impact of Ionic Liquids: Recent Advances in (Eco)toxicology and (Bio)degradability

This Review aims to integrate the most recent and pertinent data available on the (bio)degradability and toxicity of ionic liquids for global and critical analysis and on the conscious use of these compounds on a large scale thereafter. The integrated data will enable focus on the recognition of toxicophores and on the way the community has been dealing with them, with the aim to obtain greener and safer ionic liquids. Also, an update of the most recent biotic and abiotic methods developed to overcome some of these challenging issues will be presented. The review structure aims to present a potential sequence of events that can occur upon discharging ionic liquids into the environment and the potential long-term consequences.

Automated evaluation of pharmaceutically active ionic liquids' (eco)toxicity through the inhibition of human carboxylesterase and Vibrio fischeri

The toxicity of 16 pharmaceutical active ionic liquids (IL-APIs) was evaluated by automated approaches based on sequential injection analysis (SIA). The implemented bioassays were centered on the inhibition of human carboxylesterase 2 and Vibrio fischeri, in the presence of the tested compounds. The inhibitory effects were quantified by calculating the inhibitor concentration required to cause 50% of inhibition (EC50). The EC50 values demonstrated that the cetylpyridinium group was one of the most toxic cations and that the imidazolium group was the less toxic. The obtained results provide important information about the safety of the studied IL-APIs and their possible use as pharmaceutical drugs. The developed automated SIA methodologies are robust screening bioassays, and can be used as a generic tools to identify the (eco)toxicity of the structural elements of ILs, contributing to a sustainable development of drugs.

Nanoparticle-based assays in automated flow systems: A review.

Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detectors performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013.

Sequential injection fluorimetric determination of Sn in juices of canned fruits

The present work describes the development of a fast and robust sequential injection fluorimetric procedure for the determination of Sn in juices of canned fruits. The developed automatic methodology is based on the complexation of Sn with 8-hydroxyquinoline-5-sulfonic acid (HQSA) to form a fluorimetric product (lambda(exc)=354 nm; lambda(em)=510 nm). The influence of dimethylsulfoxide (DMSO) and cetylpyridinium bromide (CPB) on the sensitivity of the fluorimetric determination was evaluated. Linear calibration plots were obtained for Sn concentrations between 1 and 10 mg L(-1), with a detection limit of 0.38 mg L(-1). In each analytical cycle 0.006 mg of HQSA and 0.47 mg of CPB were consumed and 1.5 mL of effluent was generated. The developed methodology was applied to the determination of Sn in juices of canned fruits and the results complied with those furnished by an electrothermal atomic absorption spectrometry comparison procedure, with relative deviations lower than 5.2%. The automatic procedure exhibited good precision (R.S.D.<1.4%) and the sampling rate was about 70 determinations per hour.