Analytical Methods (v.8, #26)

Front cover (5161-5162).

Contents list (5163-5168).

Electrochemical nucleic acid biosensors: from fabrication to application by Aaron Tan; Candy Lim; Shui Zou; Qian Ma; Zhiqiang Gao (5169-5189).
Electrochemical biosensors have been around since the 1960s. They are among the most researched and applied biosensors. Their high portability and biocompatibility have seen them being used in the construction of in vivo biosensors. In the past three decades, their applications have been broadened from the detection of small molecules like glucose to nucleic acids and proteins. In particular, leveraging the specific base-pairing mechanism of nucleic acids, impressive progress has been achieved in the field of electrochemical nucleic acid biosensors. Moreover, various signal amplification strategies in conjunction with microfabrication technology have been extensively studied and have resulted in significant improvements in the sensitivity and multiplexing capability of electrochemical nucleic acid biosensors. In this article, the progress in the research and applications of electrochemical nucleic acid biosensors is reviewed with an emphasis on the principal components of electrochemical nucleic acid biosensors, their construction, sensing mechanism and technical applications with some discussion on challenges and perspectives.

Integrated microfluidic aptasensor for mass spectrometric detection of vasopressin in human plasma ultrafiltrate by J. Yang; J. Zhu; R. Pei; J. A. Oliver; D. W. Landry; M. N. Stojanovic; Q. Lin (5190-5196).
We present a microfluidic aptamer-based biosensor for detection of low-molecular-weight biomarkers in patient samples. Using a microfluidic device that integrates aptamer-based specific analyte extraction, isocratic elution, and detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we demonstrate rapid, sensitive and label-free detection of arginine vasopressin (AVP) in human plasma ultrafiltrate. AVP molecules in complex matrices are specifically captured by an aptamer that is immobilized on microbeads via affinity binding in a microchamber. After the removal of unbound, contaminating molecules through washing, aptamer–AVP complexes are thermally disrupted via on-chip temperature control. Released AVP molecules are eluted with purified water and transferred to a separate microchamber, and deposited onto a single spot on a MALDI plate via repeated, piezoelectrically actuated ejection, which enriches AVP molecules over the spot area. This integrated on-chip sample processing enables the quantitative detection of low-abundance AVP by MALDI-TOF mass spectrometry in a rapid and label-free manner. Our experimental results show the detection of AVP in human plasma ultrafiltrate as low as physiologically relevant picomolar concentrations via aptamer-based selective preconcentration, demonstrating the potential of our approach as a rapid (∼1 h), sensitive clinical AVP assay.

PVA-AWP/tyrosinase functionalized screen-printed electrodes for dopamine determination by M. Stoytcheva; R. Zlatev; F. F. Gonzalez Navarro; Z. Velkova; V. Gochev; G. Montero; A. G. Ayala Bautista; L. Toscano-Palomar (5197-5203).
The development of highly sensitive amperometric biosensors for dopamine determination using commercially available SPEs in combination with the PVA-AWP photopolymer as the enzyme immobilization matrix is described in this work. This research focuses on (i) the optimization of the conditions for SPE biofunctionalization by tyrosinase entrapment in PVA-AWP and of the working conditions for dopamine determination by mathematical model application, and (ii) the evaluation of the analytical performances of the PVA-AWP/tyrosinase functionalized SPEs. Dopamine determination under optimum conditions for electrode biofunctionalization (PVA-AWP 3%, 60 min UV light exposure) and under optimum working conditions (pH 6.5, 25 °C) was performed in the dynamic concentration range of 0.9–500 μmol L−1, 0.2–400 μmol L−1, and 0.03–150 μmol L−1, using respectively C SPE, MWCNT-GNP/C SPE, and graphene-GNP/C SPE. The LOD was found to be 300 nmol L−1, 60 nmol L−1, and as low as 10 nmol L−1, correspondingly. The excellent analytical performances of the graphene-GNP C SPE and MWCNT-GNP C SPE were associated with their large active area (19.72 mm2 and 15.3 mm2) and enhanced electrocatalytic properties (ko = 8.1 × 10−3 cm s−1 and 4.4 × 10−3 cm s−1) compared with the bare C SPE (12.47 mm2 and ko = 3.0 × 10−3 cm s−1). The developed biosensors were stable, reproducible, and more sensitive than most of the known biosensors for dopamine determination. They were successfully applied for dopamine determination in injections. Taking into consideration the fact that the selected enzyme immobilization approach using a water-soluble photopolymer opens the possibility of SPE biofunctionalization by photolithography, the developed biosensors are promising for fast, simple, sensitive, selective, and cost effective analysis of dopamine.

Comparison of three indirect immunoassay formats on a common paper-based microfluidic device architecture by Syrena C. Fernandes; Gabriel S. Logounov; James B. Munro; Charles R. Mace (5204-5211).
The ability to detect antibodies that are generated during an immune response is integral to the diagnosis and monitoring of infections. Assays that have been applied to the detection of antibodies are classically referred to as indirect immunoassays, which include three different formats: indirect, double-antigen sandwich, and total IgG capture. Each of these three approaches relies on a unique pair of reagents to capture the target antibody and transduce a detectable signal, which permits assays to be tuned for ideal performance based on the availability and quality of reagents, the resources available to the operator, and the complexity of the sample (e.g., serum vs. saliva). This flexibility, however, can complicate the development of diagnostic tests and increase the complexity of the devices required to perform them. A general platform that can enable the rapid development of inexpensive and simple diagnostic assays would be highly desirable, particularly for those assays intended for use in limited-resource settings. In this manuscript, we describe a paper-based microfluidic device architecture that is capable of supporting these three indirect immunoassay formats by simply changing reagents and without requiring significant alterations to the design or manufacture of the device. We compare the performances of these indirect immunoassays by developing assays for HIV antibodies in human serum. These results suggest that this device format has broad potential for the development of paper-based immunoassays.

Determination of cocaine and its main adulterants in seized drugs from Rio Grande do Sul, Brazil, by a Doehlert optimized LC-DAD method by M. C. A. Marcelo; T. R. Fiorentin; K. C. Mariotti; R. S. Ortiz; R. P. Limberger; M. F. Ferrão (5212-5217).
The main reason for the increasing cocaine consumption in South America is the high consumption of drugs in Brazil, which is the largest market on the continent. In light of this, the Brazilian Federal Police (BFP) started implementing its own drug chemical profiling program, the PeQui project, aiming to provide useful technical-scientific information about the drug scenario in the country. In this article, a liquid chromatography with a diode array detector (LC-DAD) method was developed through Doehlert optimization for the analysis of cocaine seized in the state of Rio Grande do Sul, Brazil, by the Brazilian Federal Police. In addition to cocaine, the main cocaine adulterants (diltiazem, benzocaine, levamisole, caffeine, phenacetin, lidocaine and dipyrone) were also evaluated. Through Doehlert optimization relating to the resolution and total area, a mobile phase consisting of acetonitrile : water (isocratic mode) with phosphate buffer (pH 8.3) was chosen. Fifty eight cocaine samples seized in 2013–2015 were analyzed. The average cocaine content was 45% of the drug weight and the only adulterants detected were levamisole, phenacetin and caffeine. Levamisole was detected only in salt cocaine samples and low concentrations (below 0.1 mg g−1), whereas phenacetin was detected in base form cocaine in higher concentrations. Caffeine was the only adulterant detected in both the salt and base forms, and was also at low concentrations. These results showed that the drugs seized in this Brazilian state had, on average, a lower cocaine content in relation to the rest of the country.

A novel, simple and efficient sample preparation procedure was established for the fast extraction and preconcentration of six trace components, daphnetin, scopoletin, umbelliferone, daphnoretin, isopimpinellin and ebracteolata compound B, in a traditional Chinese drug Langdu preparation using a combination of microwave-assisted extraction (MAE) and ionic liquid-based dispersive liquid–liquid microextraction (IL-based DLLME) coupled with high-performance liquid chromatography (HPLC). Several important parameters affecting extraction performance, such as microwave power, temperature and irradiation time; type and volume of extraction solvent; type and volume of dispersion solvent; centrifugation speed and time; pH of sample solution and salt concentration, were evaluated. Under optimal experimental conditions, the method exhibited a good performance in terms of linearity with correlation coefficients over 0.9993 and limits of detection (LODs) within the range 0.28–0.56 ng mL−1. The present method was applied to determine the above six analytes in a Langdu preparation named the ‘Jieheling’ tablet and the extraction recoveries obtained were in the range of 80.1–92.9%. The results indicated that the proposed method is an accurate, rapid and reliable technique, which gives excellent enrichment factors (EFs) and LODs for the extraction and preconcentration of trace analytes from a complex matrix.

Child safety: a validated GC-MS method to determine formamide in EVA foam mats by W. Wollinger; T. M. Monteiro; E. G. Campos; E. C. P. do Rego; V. S. da Cunha (5228-5233).
Recent studies on EVA products sold in Europe and manufactured in Asia have shown that puzzle mats and other EVA toys contained up to 1266 mg kg−1 of formamide, a substance classified as carcinogenic, mutagenic or toxic for reproduction. Considering this risk, France has restricted formamide in mats to no more than 200 mg kg−1, while the European Commission accepts 5000 mg kg−1. Since there is no information regarding the formamide content in EVA mats from the Brazilian market, this study aimed to first develop and validate a method for formamide determination, then analyze the best-selling EVA foam mats produced and sold in Brazil. Formamide was ultrasound-extracted three times with methanol to ensure maximum extraction. After the addition of acetamide as the internal standard, the samples were analyzed by GC-MS. This method was fully validated and presented a limit of quantification of 0.19 mg kg−1 of formamide in the chromatographed extract, recovery between 88 and 101% and RSD below 3% for concentration levels higher than 1.5 mg kg−1. Five commercial foam EVA puzzle mats made in Brazil were analyzed and presented 32 to 1364 mg kg−1 of formamide. Subsequent tests showed that usually more than 50% of the formamide amount is released and could be inhaled from the mats in the first two weeks after opening the package. Considering the exposure to formamide demonstrated in this work, we recommend that the Brazilian regulation for toy safety includes a limit for formamide content in EVA foam toys.

A new electrochemical biosensor based on the hybridization chain reaction and layered molybdenum selenide (MoSe2) stacked nanorod for dual-signal amplification is developed for the highly sensitive detection of the DNA sequences of Human Immunodeficiency Virus type 1 (HIV-1). The MoSe2 nanorod was prepared using a facile hydrothermal method. The glassy carbon electrode was first modified with MoSe2 nanorods and then Au nanoparticles (AuNPs) were electrodeposited on the electrode. The DNA probe was then immobilized on the electrode by an Au–S bond. As the initiator strands, auxiliary DNA can propagate a chain reaction of hybridization events between the two hairpin probes (Aux 1, Aux 2), and long nicked DNA polymers can be formed on the modified electrode. The biotin-labeled dsDNA polymers can then introduce numerous avidin-labeled horseradish peroxidases on the electrode, resulting in a significantly amplified electrochemical signal through the electrocatalysis of horseradish peroxidase. The target DNA is detected on the basis of the enzymatic oxidization of Fe2+ by H2O2. The electrochemical signals have a good linear relationship with the logarithm of target DNA concentration in the range from 10 fM to 0.1 pM, reaching a detection limit of the target DNA as low as 4.0 fM. This method shows good specificity for target DNA detection with the capability to discriminate single-base-pair mismatched DNA mutations. Moreover, the developed strategy was applied successfully in a real sample, which demonstrated the potential applications in clinical diagnostics.

In this work, a novel, simple and label-free electrochemiluminescence (ECL) strategy was designed for a sensitive carcinoembryonic antigen (CEA) assay on the basis of ZnS–CdS nanoparticle (NP)-decorated molybdenum disulfide (MoS2) nanocomposites. Layered-MoS2 nanocomposites with a large surface area were prepared with a facile hydrothermal method and used as an electrode matrix for subsequent ZnS–CdS NP electrodeposition. The ZnS–CdS NP decorated MoS2 electrode shows a higher ECL intensity than an electrode with only ZnS–CdS electrodeposition. For the fabrication of the aptasensor, chitosan was coated on a ZnS–CdS/MoS2/glass carbon electrode for CEA aptamer immobilization through a covalent coupling method. The ECL aptasensor for CEA detection exhibits a linear range from 0.05 to 20 ng mL−1 (R = 0.9975) with a low detection limit of 0.031 ng mL−1, and presents satisfactory specificity against some possible interfering proteins. Moreover, the method was successfully applied for CEA determination in human serum with recoveries of 80–111%, showing great promise for application in practice.

A sensitive and rapid method involving an improved approach called the quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification method and isotope dilution gas chromatography-mass spectrometry (GC-MS) was established to determine six N-nitrosamines (NAs) in soy sauce. Samples were firstly extracted by ethyl acetate. Then the extraction solution was concentrated by slow nitrogen gas blowing and subsequently purified using the QuEChERS-based purification method. Separation of six target NAs was performed on an INNOWAX polar capillary chromatographic column. All of the samples were detected by selected ion monitoring (SIM) mode of GC-MS and N-nitrosodimethylamine-d6 (NDMA-d6) and N-nitrosodipropylamine-d14 (NDPA-d14) were used as internal standards. The developed method was validated in terms of the linearity, limit of detection (LOD), matrix effects, specificity, accuracy and precision. Results indicated that this method is of good specificity with almost negligible matrix effects. Linear relations of six NAs were favourable in the range of 2.0–200 μg L−1 and the correlation coefficients were greater than 0.9994. The LODs were lower than 1 μg kg−1 (0.4–0.9 μg kg−1) and the limits of quantitation (LOQs) of the method were within the range of 1.2–3.0 μg kg−1. The mean recoveries for negative light and dark soy sauce samples at three spiked concentration levels were in the range of 80.2–112% with overall relative standard deviation (RSD) values (n = 6) of 2.5–6.8%. The validated method was simple and rapid, with good repeatability, and was successfully applied to determine the concentrations of NAs in soy sauce.

Targeted histidine-peptide enrichment improved the accuracy of isobaric-based quantitative proteomics by Jingjing Yang; Fang Tian; Manli Zhang; Yang Zhao; Xiaohong Qian; Yun Cai; Wantao Ying (5255-5261).
Liquid chromatography combined with tandem mass spectrometry has been widely used for in-depth proteome profiling and to quantitatively measure proteome regulation. However, due to peak capacity constraints, differences in peptide properties and other factors in total proteome analysis, only limited protein sequence coverage can be obtained. A large number of co-eluted peptides may act as a matrix and co-fragment with the targeted parent ions, seriously affecting the accuracy of fragment ion-based quantitative proteomic analysis. To simplify the complicated peptide mixture and improve the accuracy of the quantitative mass spectrometric analysis, this research explored a method for selective enrichment of peptides containing histidine (his-peptides). The experimental conditions, including Cu-bead preparation, the binding duration of his-peptides to the beads, and the ratio of the total peptide mass to volume of the beads, were evaluated. The optimized procedure for his-peptide enrichment was fast and robust and provided 70% recovery of the his-peptides in a highly reproducible way. When combined with an exemplary iTRAQ experiment, the his-peptide enrichment approach can significantly improve the accuracy of the quantitative analysis. Therefore, his-peptide enrichment can be used as a simple, effective strategy to simplify the complex proteome and improve the accuracy of quantitative proteome research.

A greener flow injection method based on a LWCC for the screening of tetracycline antibiotics in bovine milk samples by Michael Pérez Rodríguez; Bianca Ferreira da Silva; Helena Redigolo Pezza; Leonardo Pezza (5262-5271).
An easy and greener flow injection method was developed for the spectrophotometric screening of tetracycline antibiotic residues in bovine milk. The combined benefits of the use of a detection system based on a liquid waveguide capillary cell (LWCC) and stop-flow (60 s) conditions significantly enhanced (45.6-fold) the analytical sensitivity. The determination is based on the formation of tetracycline azo derivatives (λmax = 435 nm) from diazotized p-sulfanilic acid, in a slightly alkaline medium. The analytical conditions were optimized by multivariate analysis, using a fractional factorial design. The linear range was 50–400 μg L−1, and the limits of detection and quantification were in the ranges 8.6–12.2 μg L−1 and 28.8–40.7 μg L−1, respectively. The applicability of the LWCC-based method was successfully tested using fortified milk samples that were only submitted to a prior acid treatment. The screening provided a quick response, enabling the determination of whether the tetracyclines were above or below the limit established by legislation (100 μg L−1). The positive samples were also analyzed by an AOAC Official Method, and the results obtained with the two methods were in good agreement. Additionally, the presence of tetracycline residues was confirmed using an HPLC-MS/MS reference analytical procedure. The excellent performance of the new screening method makes it suitable for use in the quality control of bovine milk in the food industry, aiming at consumer protection.

A comparison of techniques for size measurement of nanoparticles in cell culture medium by Christian Gollwitzer; Dorota Bartczak; Heidi Goenaga-Infante; Vikram Kestens; Michael Krumrey; Caterina Minelli; Marcell Pálmai; Yannic Ramaye; Gert Roebben; Aneta Sikora; Zoltán Varga (5272-5282).
Plain and aminated silica nanoparticles dispersed in purified water, in 50 mM Tris–HCl buffer and in cell culture medium were measured using dynamic light scattering (DLS), centrifugal liquid sedimentation (CLS), small-angle X-ray scattering (SAXS), and particle tracking analysis (PTA). The test samples were measured by all methods immediately after dispersion and after incubation at room temperature for 24 h. The effect of the biological dispersion medium on the modal value of the particle size distribution was compared for each method taking into account the estimated uncertainty. For the methods based on light scattering, DLS and PTA, the size distributions obtained were significantly altered due to the formation of a protein corona and induced agglomeration effects. With SAXS and CLS, the measured size of the primary particles was mostly unchanged. While SAXS offers excellent precision and traceability to the SI unit system if the model fitting approach is used for data analysis, CLS provides detailed size distributions from which additional information on the agglomeration state can be deduced.

A selective and low organic-solvent-consuming method of sample preparation combined with high-performance liquid chromatography-mass spectrometry detection is introduced for the analysis of α-tocopherol in cereal grains. The sample treatment involves initial saponification and preconcentration of the analyte performed by ionic-liquid-based dispersive liquid–liquid microextraction, with ethanol as disperser solvent and 1-octyl-3-methylimidazolium hexafluorophosphate as extraction solvent. The method offered excellent linearity over a range of 0.5–15 μg g−1 with a correlation coefficient of r > 0.998. Intra-day and inter-day repeatability, expressed as relative standard deviation were 3.4% and 5.9%, respectively. The quantitation limits and detection limits were 2.5 and 0.7 ng g−1. Satisfactory recoveries ranging from 86.5% to 106.3% were obtained. The developed method was successfully applied to 12 cereal grain samples.

In this study, a new type of organophosphate pesticide (OP) biosensor was successfully fabricated based on the immobilization of acetylcholinesterase (AChE) by cross-linking on a glassy carbon electrode (GCE) modified with ionic liquid functionalized graphene (IL-GR), Co3O4 nanoparticles and chitosan (CHI). The introduction of IL-GR and Co3O4 nanoparticles not only enhanced the surface area of the modified electrode for enzyme immobilization but also facilitated the electron transfer, resulting in a high sensitivity of the biosensor. The fabrication process of the sensing surface was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). For the oxidation of thiocholine, a hydrolysis product of acetylthiocholine, the peak current at the AChE/IL-GR/Co3O4/CHI/GCE electrode is larger than those at AChE/IL-GR/CHI/GCE and AChE/Co3O4/CHI/GCE electrodes. A linear relationship between the inhibition percentage (I%) and logarithm of the concentration of dimethoate was found in the range from 5.0 × 10−12 to 1.0 × 10−7 M, with a detection limit of 1.0 × 10−13 M (S/N = 3). The proposed biosensor provided an efficient and promising platform for the immobilization of AChE and exhibited higher sensitivity and acceptable stability for the detection of organophosphate pesticides.

Magnetic dispersive solid-phase extraction based on a novel adsorbent for the detection of triazole pesticide residues in honey by HPLC-MS/MS by Qiongchen Miao; Jianmei Wang; Jing Nie; Huizhen Wu; Yanping Liu; Zuguang Li; Mingrong Qian (5296-5303).
A novel Fe3O4@MDN magnetic nanomaterial was successfully developed as a magnetic dispersive solid-phase extraction adsorbent for the determination of triazole pesticide residuals in honey samples by high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-MS/MS). The macromolecular structure named MDN was coated on the surface of a copolymer synthesized with silyl reagents methacrylic acid-3-(trimethoxysilyl) propyl ester (MPS) and tetraethyl orthosilicate (TEOS) based on ferriferrous oxide (Fe3O4), whose characteristics were confirmed by a scanning electron microscope (SEM). The proposed magnetic dispersive solid-phase extraction (MDSPE) method provided a rapid, environmentally friendly and magnetic stuff recyclable approach for the triazole pesticide analysis. By means of optimal conditions, the recoveries of 15 triazoles (spiked at 20 μg kg−1) ranged from 90.5 to 105.9%. The method showed good linearity, sensitivity in the range of μg per kilogram and repeatability (relative standard deviation below 6.8%, n = 5). The proposed method was confirmed to be a simple, rapid and efficient method for the analysis of triazole pesticide residuals in honey samples.

The paper presents the results of a study on the efficiency of the separation of Ta(V) and Nb(V) from fluoride solutions by solvent extraction with methyl isobutyl ketone with a view to applying the extraction process to the purification of Nb from Ta and for the pre-concentration of Ta prior to its determination in Nb and its compounds over a wide range of initial concentrations. With strict control of the hydrofluoric acid concentration and the re-extraction of Ta into an aqueous phase using an ammonium oxalate solution, the mean recovery factors R̄[%] for Ta were 94–108% in the initial weight ratio mNb : mTa = (ρNb/Ta) range 10–1 000 000 with only a small scatter about the mean value (RSD ≤ 6%), with the exception of (ρNb/Ta) = 1 000 000. The effect of the composition of the re-extraction solution on the excitation conditions of Ta in ICP-OES (ICP-MS) plasma was negligible and small amounts of Nb and other contaminants co-extracted with Ta did not affect its determination.

Back cover (5311-5312).