Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds.

The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme ß-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-ß-d-galactopyranoside, is proposed, allowing for the assessment of EDCs' presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17ß-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.

Exploiting Kinetic Features of ORAC Assay for Evaluation of Radical Scavenging Capacity.

The analysis and interpretation of data retrieved from Oxygen Radical Absorbance Capacity (ORAC) assays represent a challenging task. ORAC indexes originate from different mathematical approaches often lacking correct elucidation of kinetic features concerning radical scavenging reactions by antioxidant compounds. In this work, the expression of ORAC values as area under fluorescein (FL) decay curves (AUC) and lag time are critically compared. This multi-parametric analysis showed the extension of radical scavenging reactions beyond the lag time period for caffeic acid, gallic acid, reduced glutathione and quercetin, extending their antioxidant protection of FL. Ethanol delayed the reaction of both FL and antioxidant compounds with free radical species generated from 2,2'-azobis(2-amidinopropane) dihydrochloride thermolysis. Trolox equivalent values, commonly used to express ORAC values, were more affected by the differences in radical scavenging kinetics between the reference and the tested antioxidant compounds when calculated from AUC than from lag time. These findings stressed the importance of choosing calibrator compounds presenting ORAC kinetics similar to samples to prevent biased estimation of the antioxidant capacity. Additionally, the framework proposed here provides a sustainable analytical method for the evaluation of antioxidant capacity, with an AGREE score of 0.73.

Lab-on-Valve Automated and Miniaturized Assessment of Nanoparticle Concentration Based on Light-Scattering.

Nanoparticles (NPs) concentration directly impacts the dose delivered to target tissues by nanocarriers. The evaluation of this parameter is required during NPs developmental and quality control stages, for setting dose-response correlations and for evaluating the reproducibility of the manufacturing process. Still, faster and simpler procedures, dismissing skilled operators and post-analysis conversions are needed to quantify NPs for research and quality control operations, and to support result validation. Herein, a miniaturized automated ensemble method to measure NPs concentration was established under the lab-on-valve (LOV) mesofluidic platform. Automatic NPs sampling and delivery to the LOV detection unit were set by flow programming. NPs concentration measurements were based on the decrease in the light transmitted to the detector due to the light scattered by NPs when passing through the optical path. Each analysis was accomplished in 2 min, rendering a determination throughput of 30 h-1 (6 samples h-1 for n = 5) and only requiring 30 µL (≈0.03 g) of NPs suspension. Measurements were performed on polymeric NPs, as these represent one of the major classes of NPs under development for drug-delivery aims. Determinations for polystyrene NPs (of 100, 200, and 500 nm) and for NPs made of PEGylated poly-d,l-lactide-co-glycolide (PEG-PLGA, a biocompatible FDA-approved polymer) were accomplished within 108-1012 particles mL-1 range, depending on the NPs size and composition. NPs size and concentration were maintained during analysis, as verified for NPs eluted from the LOV by particle tracking analysis (PTA). Moreover, concentration measurements for PEG-PLGA NPs loaded with an anti-inflammatory drug, methotrexate (MTX), after their incubation in simulated gastric and intestinal fluids were successfully achieved (recovery values of 102-115%, as confirmed by PTA), showing the suitability of the proposed method to support the development of polymeric NPs targeting intestinal delivery.

Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers.

The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.

Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection.

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 µg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 µL) from serum and saliva.

Automated lab-on-valve sequential injection ELISA for determination of carbamazepine.

The development of an automated miniaturized analytical system that allows for the rapid monitoring of carbamazepine (CBZ) levels in serum and wastewater is proposed. Molecular recognition of CBZ was achieved through its selective interaction with microbeads carrying anti-CBZ antibodies. The proposed method combines the advantages of the micro-bead injection spectroscopy and of the flow-based platform lab-on-valve for implementation of automatic immunosorbent renewal, rendering a new recognition surface for each sample. The sequential (or simultaneous) perfusion of CBZ and the horseradish peroxidase-labelled CBZ through the microbeads is followed by real-time on-column monitoring of substrate (3,3',5,5'-tetramethylbenzidine) oxidation by colorimetry. The evaluation of the initial oxidation rate and also the absorbance value at a fixed time point provided a linear response versus the logarithm of the CBZ concentration. Under the selected assay conditions, a single analysis was completed after only 11 min, with a quantification range between 1.0 and 50 µg L-1. Detection of CBZ levels in undiluted wastewater samples was feasible after a simple filtration step while good recoveries were attained for spiked certified human serum, analyzed without sample clean-up.

Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure.

Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagents are expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via N-hydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum.

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (µ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL-1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 µg mL-1 (corresponding to 5.0-60 mg mL-1 in undiluted samples), with a detection limit of 33 µg mL-1 (1.7 mg mL-1 for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

Chiral Derivatives of Xanthones: Investigation of the Effect of Enantioselectivity on Inhibition of Cyclooxygenases (COX-1 and COX-2) and Binding Interaction with Human Serum Albumin.

Searching of new enantiomerically pure chiral derivatives of xanthones (CDXs) with potential pharmacological properties, particularly those with anti-inflammatory activity, has remained an area of interest of our group. Herein, we describe in silico studies and in vitro inhibitory assays of cyclooxygenases (COX-1 and COX-2) for different enantiomeric pairs of CDXs. The evaluation of the inhibitory activities was performed by using the COX Inhibitor Screening Assay Kit. Docking simulations between the small molecules (CDXs; known ligands and decoys) and the enzyme targets were undertaken with AutoDock Vina embedded in PyRx-Virtual Screening Tool software. All the CDXs evaluated exhibited COX-1 and COX-2 inhibition potential as predicted. Considering that the (S)-(-)-enantiomer of the nonsteroidal anti-inflammatory drug ketoprofen preferentially binds to albumin, resulting in lower free plasma concentration than (R)-(+)-enantiomer, protein binding affinity for CDXs was also evaluated by spectrofluorimetry as well as in in silico. For some CDXs enantioselectivity was observed.

Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 µM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste.

On-line automated evaluation of lipid nanoparticles transdermal permeation using Franz diffusion cell and low-pressure chromatography.

A low-pressure liquid chromatography system for the on-line quantification of caffeine loaded into lipid nanoparticles that permeates pig skin was developed. The apparatus includes a Franz diffusion cell with computer-controlled sampling that allows collection of acceptor solution with automatic compensation for sample withdrawing, and a C-18 reversed-phase monolithic column integrated in a typical Flow Injection Analysis (FIA) set-up where separation between caffeine and other matrix elements is performed before spectrophotometric quantification at 273 nm. Several parameters regarding chromatographic analysis (propulsion element, column length, mobile phase composition, and flow rate) were studied along with the establishment of the sampling procedure. Under the selected conditions (monolithic column Chromolith® RP-18 15 mm × 4.6 mm i.d., acetonitrile:water 10:90 (v/v), flow rate 0.45 mL min(-1)) a detection limit of 4 µM and RSD values for caffeine concentration <2% were achieved. High recovery values were obtained when Hepes buffer incubated as acceptor solution in presence of pig skin for 8 h was spiked with caffeine (103±5%). The developed system also accounts for low organic solvent consumption, low operating costs, low generation of waste and high sample throughput (24 h(-1)). Due to the real time automated sampling and high throughput, transdermal permeation profiles of nanoformulations can be established within a time frame seldom observed by conventional techniques.