Analytica Chimica Acta (v.759, #C)

Polyaniline coated micro-capillaries for continuous flow analysis of aqueous solutions by Larisa Florea; Dermot Diamond; Fernando Benito-Lopez (1-7).
Display Omitted► Microcapillaries were coated with polyaniline nanofibres by the grafting approach. ► Optical detection of aqueous ammonia in continuous flow mode was achieved. ► The sensing platform can be easily regenerated after detection. ► Very small volumes of analytes are necessary for detection. ► The nanostructure of the coating guarantees fast response and regeneration times.The inner walls of fused silica micro-capillaries were successfully coated with polyaniline nanofibres using the “grafting” approach. The optical response of polyaniline coatings was evaluated during the subsequent redoping–dedoping processes with hydrochloric acid and ammonia solutions, respectively, that were passed inside the micro-capillary in continuous flow. The optical absorbance of the polyaniline coatings was measured and analysed in the wavelength interval of [300–850 nm] to determine its optical sensitivity to different concentrations of ammonia. It was found that the optical properties of polyaniline coatings change in response to ammonia solutions in a wide concentration range from 0.2 ppm to 2000 ppm. The polyaniline coatings employed as a sensing material for the optical detection of aqueous ammonia have a fast response time and a fast regeneration time of less than 5 s at room temperature. The coating was fully characterised by scanning electron microscopy, Raman spectroscopy, absorbance measurements and kinetic studies. The response of the coatings showed very good reproducibility, demonstrating that this platform can be used for the development of micro-capillary integrated sensors based on the inherited sensing properties of polyaniline.
Keywords: Polyaniline; Continuous flow; Sensor; Ammonia; Micro-capillary;

Display Omitted► Multivariate curve resolution (MCR) applied to water spectra at different temperatures and salt content. ► Three distinct components with varying temperature dependence were present in water perturbed by temperature. ► Locations of their MCR pure components of salt solutions varied according to the ionic strength of the salt used.Aquaphotomics is a new discipline that provides a framework for understanding changes in the structure of water caused by various perturbations, such as variations in temperature or the addition of solutes, using near infrared spectroscopy (NIRS). One of the main purposes of aquaphotomics is to identify water bands as main coordinates of future absorbance patterns to be used as biomarkers. These bands appear as consequence of perturbations in the NIR spectra. Curve resolution techniques may help to resolve and find new water bands or confirm already known bands. The aim of this study is to investigate the application of multivariate curve resolution-alternating least squares (MCR-ALS) to characterise the effects of various perturbations on the NIR spectra of water in terms of hydrogen bonding. For this purpose, the perturbations created by temperature change and the addition of four solutions of different ionic strength and Lewis acidity were studied (NaCl, KCl, MgCl2 and AlCl3, with concentrations ranging from 0.2 to 1 mol L−1 in steps of 0.2 mol L−1). Transmission spectra of all salt solutions and pure water were obtained at temperatures ranging from 28 to 45 °C. We have found that three distinct components with varying temperature dependence are present in water perturbed by temperature. The salt solutions studied exhibited similar trends with respect to the temperature perturbation, while the peak locations of their MCR-ALS pure components varied according to the ionic strength of the salt used.
Keywords: Water; Near infrared; Hydrogen bond; Multivariate curve resolution; Aquaphotomics;

Display Omitted► Classification of ToF-SIMS mass spectra from complex Cu–Fe sulphides was achieved. ► Supervised chemometrics methods, ANN and PCA-ANN were used for classification. ► Improved information and classification were obtained from the PCA-ANN classifier.Artificial neural network (ANN) and a hybrid principal component analysis-artificial neural network (PCA-ANN) classifiers have been successfully implemented for classification of static time-of-flight secondary ion mass spectrometry (ToF-SIMS) mass spectra collected from complex Cu–Fe sulphides (chalcopyrite, bornite, chalcocite and pyrite) at different flotation conditions. ANNs are very good pattern classifiers because of: their ability to learn and generalise patterns that are not linearly separable; their fault and noise tolerance capability; and high parallelism. In the first approach, fragments from the whole ToF-SIMS spectrum were used as input to the ANN, the model yielded high overall correct classification rates of 100% for feed samples, 88% for conditioned feed samples and 91% for Eh modified samples. In the second approach, the hybrid pattern classifier PCA-ANN was integrated. PCA is a very effective multivariate data analysis tool applied to enhance species features and reduce data dimensionality. Principal component (PC) scores which accounted for 95% of the raw spectral data variance, were used as input to the ANN, the model yielded high overall correct classification rates of 88% for conditioned feed samples and 95% for Eh modified samples.
Keywords: Time-of-flight secondary ion mass spectrometry; Cu–Fe sulphides; Flotation; Principle component analysis; Artificial neural networks;

Display Omitted► The concept of applicability domain (AD) in QSAR modeling is discussed. ► The AD assessment method for nonlinear neural network predictive models is proposed. ► The counter-propagation artificial neural network (CP-ANN) was applied for modeling. ► Minimal Euclidean distance space (MEDS) of CP-ANN model was defined and analyzed. ► The resulting outliers coincide with those from linear models (leverage based AD).Alongside the validation, the concept of applicability domain (AD) is probably one of the most important aspects which determine the quality as well as reliability of the established quantitative structure–activity relationship (QSAR) models. To date, a variety of approaches for AD estimation have been devised which can be applied to particular type of QSAR models and their practical utilization is extensively elaborated in the literature. The present study introduces a novel, simple, and effective distance-based method for estimation of the AD in case of developed and validated predictive counter-propagation artificial neural network (CP ANN) models through a proficient exploitation of the Euclidean distance (ED) metric in the structure-representation vector space. The performance of the method was evaluated and explained in a case study by using a pre-built and validated CP ANN model for prediction of the transport activity of the transmembrane protein bilitranslocase for a diverse set of compounds. The method was tested on two more datasets in order to confirm its performance for evaluation of the applicability domain in CP ANN models. The chemical compounds determined as potential outliers, i.e., outside of the CP ANN model AD, were confirmed in a comparative AD assessment by using the leverage approach. Moreover, the method offers a graphical depiction of the AD for fast and simple determination of the extreme points.
Keywords: Quantitative structure–activity relationship; Artificial neural networks; Counter-propagation artificial neural network; Applicability domain; Euclidean distance; Leverage approach;

Display Omitted► The QSAR model is not dependent of ligand conformation. ► Amphetamines were analyzed by quantum chemical, steric and hydrophobic descriptors. ► CHELPG atomic charges on the benzene ring are one of the most important descriptors. ► The PLS models built were extensively validated. ► Manual docking supports the QSAR results by pi–pi stacking interactions.Chiral molecules need special attention in drug design. In this sense, the R and S configurations of a series of thirty-four amphetamines were evaluated by quantitative structure–activity relationship (QSAR). This class of compounds has antidepressant, anti-Parkinson and anti-Alzheimer effects against the enzyme monoamine oxidase A (MAO A). A set of thirty-eight descriptors, including electronic, steric and hydrophobic ones, were calculated. Variable selection was performed through the correlation coefficients followed by the ordered predictor selection (OPS) algorithm. Six descriptors (CHELPG atomic charges C3, C4 and C5, electrophilicity, molecular surface area and log  P) were selected for both configurations and a satisfactory model was obtained by PLS regression with three latent variables with R 2  = 0.73 and Q 2  = 0.60, with external predictability Q 2  = 0.68, and R 2  = 0.76 and Q 2  = 0.67 with external predictability Q 2  = 0.50, for R and S configurations, respectively. To confirm the robustness of each model, leave-N-out cross validation (LNO) was carried out and the y-randomization test was used to check if these models present chance correlation. Moreover, both automated or a manual molecular docking indicate that the reaction of ligands with the enzyme occurs via pi–pi stacking interaction with Tyr407, inclined face-to-face interaction with Tyr444, while aromatic hydrogen–hydrogen interactions with Tyr197 are preferable for R instead of S configurations.
Keywords: Quantitative structure–activity relationship; Amphetamine derivatives; Electronic; Hydrophobic and steric descriptors; R and S configurations of amphetamines;

Display Omitted► A novel apoferritin nanoparticles modified with HRP and aptamer was synthesized. ► Signal dual amplification was carried out by using the resulted nanoparticles as detection probe. ► Ultrasensitive and high specific thrombin detection was achieved.A novel and ultrasensitive sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Core/shell Fe3O4/Au magnetic nanoparticles (AuMNPs) loading aptamer1 (Apt1) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5–100 pM with a detection limit of 0.07 pM (S/N  = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in protein detection and disease diagnosis.
Keywords: Apoferritin; Thrombin; Horseradish peroxidase; Electrochemical aptasensor;

Display Omitted► Direct electrochemistry of glucose oxidase used for signal generation in aptasensor. ► Using novel nanocomposite for immobilization and signal amplification. ► Sensitive electrochemical detection of platelet-derived growth factor.In this work, a new label-free electrochemical aptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growth factor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). For this proposed aptasensor, poly(diallyldimethylammonium chloride) (PDDA)-protected graphene-gold nanoparticles (P-Gra-GNPs) composite was firstly coated on electrode surface to form the interface with biocompatibility and huge surface area for the adsorption of GOD layer. Subsequently, gold nanoclusters (GNCs) were deposited on the surface of GOD to capture PDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to block the remaining active sites of the GNCs and avoid the nonspecific adsorption. With the direct electron transfer of double layer GOD membranes, the aptasensor showed excellent electrochemical response and the peak current decreased linearly with increasing logarithm of PDGF concentration from 0.005 nM to 60 nM with a relatively low limit of detection of 1.7 pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-term stability, which provided a new promising technique for aptamer-based protein detection.
Keywords: Aptasensor; Glucose oxidase; Direct electrochemistry; Graphene; Platelet-derived growth factor;

Display Omitted► A SPME–GC–QqQ MS approach was developed for the quantification of acidic biomarkers in human urine. ► The combined use of alkylchloroformate and SPME allows one to minimize the presence of organic solvents in the final protocol. ► The specificity of the MRM acquisition mode improves the ability in analytes identification.Homovanillic acid (HVA), vanylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA) are the metabolites of some catecholamines such as epinephrine, nor-epinephrine, dopamine and serotonin and their quantification is used in the diagnosis and management of patients with neurocrine tumors. A novel approach in the assay of these biomarkers in human urine samples by solid phase microextraction (SPME) combined with gas chromatography-triple quadrupole mass spectrometry (GC–QqQ-MS) is presented. A preliminary derivatization with ethyl chloroformate/ethanol was used and the corresponding derivatives were then extracted by SPME in immersion mode. The performance of five SPME fibers and three chloroformates were evaluated in univariate mode and the best results were obtained using the polyacrylate fiber and ethyl chloroformate. The variables affecting the efficiency of SPME analysis were optimized by the multivariate approach of “Experimental design” and, in particular, a central composite design (CCD) was applied. The optimum working conditions in terms of response values were achieved by performing analysis at room temperature with addition of NaCl (9.5%) and with an extraction time of 25.8 min. Identification and quantification of analytes were carried out by using a gas chromatography-triple quadrupole mass spectrometry (GC–QqQ MS) system in multiple reaction monitoring (MRM) acquisition. An evaluation of all analytical parameters shows that the proposed method provides satisfactory results. Very good linearities were, in fact, achieved in the tested calibration ranges with correlation coefficient values >0.99 for all the analytes and accuracies and RSDs calculated for between-run and tested at concentrations of 1, 10, and 80 mg L−1 were ranging from 91.3% to 106.6%, and from 0.5 to 8.9%, respectively. Moreover, the LOD values obtained can be considered very satisfactory (1.3, 0.046 and 24.3 μg L−1 for HVA, VMA and 5-HIAA, respectively). The developed protocol represents, therefore, a simple, rapid and selective tool for assaying these acidic biomarkers in urine samples for neuroendocrine cancer diagnosis.
Keywords: Solid phase microextraction; Gas chromatography; Tandem mass spectrometry; Vanylmandelic acid; Homovanillic acid; 5-Hydroxyindoleacetic acid;

Label-free electrochemical monitoring of vasopressin in aptamer-based microfluidic biosensors by Peng He; Vlad Oncescu; Seoho Lee; Inhee Choi; David Erickson (74-80).
Vasopressin is an indicating biomarker for blood pressure in the human body and low vasopressin levels can be indicative of late-phase hemorrhagic shock or other traumatic injuries. In this paper we have developed an aptamer-based label-free microfluidic biosensor for the electrochemical detection of vasopressin. The detection area consists of aptamers immobilized on carbon nanotubes which specifically capture the vasopressin molecules in solution resulting in changes in conductivity across the sensor. We report a limit of detection of 43 pM in standard solutions and demonstrate high detection specificity toward vasopressin when different interferents are present. The miniaturized microfluidic biosensor offers continuous monitoring of different vasopressin levels with good potential for portability. Ultimately such a system could serve as a point-of-care diagnostics tool for patients with excessive bleeding when standard medical infrastructure is not available.
Keywords: Biosensor; Aptamer; Vasopressin; Carbon nanotube;

Luminescence recognition of different organophosphorus pesticides by the luminescent Eu(III)–pyridine-2,6-dicarboxylic acid probe by Hassan A. Azab; Axel Duerkop; Z.M. Anwar; Belal H.M. Hussein; Moustafa A. Rizk; Tarek Amin (81-91).
Display Omitted► Europium (III) luminescence quenching has been used for sensing organophosphorous pesticides. ► Four guest pesticides chlorfenvinphos, malathion, azinphos, and paraxon ethyl were used. ► A sensitive rapid, cheap direct method for the determination of the pesticides has been developed. ► The method was applied to the determination of the OPs in tap, river, mineral, and waste waters.Luminescence quenching of a novel long lived Eu(III)–pyridine-2,6-dicarboxylic acid probe of 1:2 stoichiometric ratio has been studied in 0.10 volume fraction ethanol–water mixture at pH 7.5 (HEPES buffer) in the presence of the organophosphorus pesticides chlorfenvinphos (P1), malathion (P2), azinphos (P3), and paraxon ethyl (P4). The luminescence intensity of Eu(III)–(PDCA)2 probe decreases as the concentration of the pesticide increases. It was observed that the quenching due to P3 and P4 proceeds via both diffusional and static quenching processes. Direct methods for the determination of the pesticides under investigation have been developed using the luminescence quenching of Eu(III)–pyridine-2,6-dicarboxylic acid probe in solution. The linear range for determination of the selected pesticides is 1.0–35.0 μM. The detection limits were 0.24–0.55 μM for P3, P4, and P1 and 2.5 μM for P2, respectively. The binding constants (K), and thermodynamic parameters of the OPs with Eu(III)–(PDCA)2 were evaluated. Positive and negative values of entropy (ΔS) and enthalpy (ΔH) changes for Eu(III)–(PDCA)2–P1 ternary complex were calculated. As the waters in this study do not contain the above mentioned OPs over the limit detectable by the method, a recovery study was carried out after the addition of the adequate amounts of the organophosphorus pesticides under investigation.
Keywords: Europium; Pyridine-2,6-dicarboxylic acid; Organophosphorus pesticides chlorfenvinphos, malathion, azinphos, and paraxon ethyl; Analytical detection; Luminescence quenching; Thermodynamic parameters;

A competitive displacement assay with quantum dots as fluorescence resonance energy transfer donors by Charles H. Vannoy; Lori Chong; Connie Le; Ulrich J. Krull (92-99).
Display Omitted► Demonstration of oligonucleotide displacement assay using quantum dots and FRET. ► Demonstration of a displacement assay that avoids labeling of target. ► Displacement assay targeting short strand embedded within a longer strand.The unique optoelectronic properties of semiconductor quantum dots (QDs) make them well-suited as fluorescent bioprobes for use in various biological applications. Modification of CdSe/ZnS QDs with biologically relevant molecules provides for multipotent probes that can be used for cellular labeling, bioassays, and localized optical interrogation by means of fluorescence resonance energy transfer (FRET). Herein, we demonstrate the use of red-emitting streptavidin-coated QDs (QD605) as donors in FRET to introduce a competitive displacement-based assay for the detection of oligonucleotides. Various QD–DNA bioconjugates featuring 25-mer probe sequences diagnostic of Hsp23 were prepared. The single-stranded oligonucleotide probes were hybridized to dye-labeled (Alexa Fluor 647) reporter sequences, which were provided for a FRET-sensitized emission signal due to proximity of the QD and dye. The dye-labeled sequence was designed to be partially complementary and include base-pair mismatches to facilitate displacement by a more energetically favorable, fully complementary recognition motif embedded within a 98-mer displacer sequence. Overall, this study demonstrates proof-of-concept at the nM level for competitive displacement hybridization assays in vitro by reduction of fluorescence intensity that directly correlates to the presence of oligonucleotides of interest. This work demonstrates an analytical method that could potentially be implemented for monitoring of intracellular gene expression in the future.
Keywords: Bioassay; Competitive displacement; Fluorescence resonance energy transfer; Oligonucleotides; Quantum dots;

Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene by Jinny L. Liu; Dan Zabetakis; Glendalys Acevedo-Vélez; Ellen R. Goldman; George P. Anderson (100-104).
Display Omitted► We describe a monoclonal antibody that recognizes 2,4,6-trinitrotoluene (TNT). ► An effective scFv was developed from the anti-TNT monoclonal antibody. ► The scFv provided enhanced limits of detection relative to the monoclonal.Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5 × 10−13  M and 4.8 × 10−10  M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75 °C while the scFv melts at ∼65 °C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1 μg L−1. The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT.
Keywords: Single chain antibody; Surface plasmon resonance; Circular dichroism; Competitive immunoassay; Explosive detection; MAGPIX; Magplex microspheres;

Highly sensitive detection of human cardiac myoglobin using a reverse sandwich immunoassay with a gold nanoparticle-enhanced surface plasmon resonance biosensor by Oksana V. Gnedenko; Yury V. Mezentsev; Andrey A. Molnar; Andrey V. Lisitsa; Alexis S. Ivanov; Alexander I. Archakov (105-109).
Display Omitted► A highly sensitive immunoassay for detection of cardiac myoglobin was designed. ► A gold nanoparticles were used for SPR signal amplification. ► The limit of detection of cMb in a serum sample was found to be as low as 10 pM.A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10 pM (approx. 0.18 ng mL−1), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.
Keywords: Surface plasmon resonance; Reverse sandwich immunoassay; Cardiac myoglobin; Gold nanoparticles; Signal amplification;