Analytica Chimica Acta (v.845, #C)

A strategy was developed by DNA recycling with target-triggered assembly of mutated DNAzyme and Zn2+ for electrochemical detection of DNA.Display OmittedA novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn2+ assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn2+. After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35 fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn2+-based amplification. The Zn2+ assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.
Keywords: Deoxyribonucleic acid; Amplification; Electrochemical; DNAzyme;

Display OmittedA simple and sensitive method to determine Hg2+ was developed by combining solution-cathode glow discharge atomic emission spectrometry (SCGD-AES) with flow injection (FI) based on on-line solid-phase extraction (SPE). We synthesized l-cysteine-modified mesoporous silica and packed it in an SPE microcolumn, which was experimentally determined to possess a good mercury adsorption capacity. An enrichment factor of 42 was achieved under optimized Hg2+ elution conditions, namely, an FI flow rate of 2.0 mL min−1 and an eluent comprised of 10% thiourea in 0.2 mol L−1 HNO3. The detection limit of FI–SCGD-AES was determined to be 0.75 μg L−1, and the precision of the 11 replicate Hg2+ measurements was 0.86% at a concentration of 100 μg L−1. The proposed method was validated by determining Hg2+ in certified reference materials such as human hair (GBW09101b) and stream sediment (GBW07310).
Keywords: Mercury; Separation and pre-concentration; Solution-cathode glow discharge; Mesoporous silica; Solid-phase extraction;

A smartphone metabolomics platform and its application to the assessment of cisplatin-induced kidney toxicity by Hyuknam Kwon; Jooeun Park; Yongjin An; Jaeho Sim; Sunghyouk Park (15-22).
Display OmittedThe application of smartphones to medical devices has been gaining attention in addressing accessibility and cost issues in healthcare, and the detection of medically relevant compounds has been demonstrated using customized smartphone hardware and/or software. Metabolomics, a newly rising omics field, has also spawned many medical applications but requires highly sophisticated and expensive equipment. Here, we describe a portable smartphone platform, built with readily available and affordable materials, that can perform all of the critical aspects of metabolomics. Excluding the smartphone itself, the total materials for the platform were obtained at less than US $20. For spectral data acquisition, the system utilized visible light (400–700 nm) and a built-in camera. All of the data processing, statistical analysis, and final-visualization components necessary for decision making were implemented in the smartphone platform. The platform is generally applicable as long as the analytes absorb visible light. We provide a proof-of-concept example wherein the metabolomics platform was applied to the assessment of cisplatin-induced kidney toxicity in a rat model, correctly predicting 7 out of 8 test samples.
Keywords: Smartphone; Metabolomics; Drug toxicity; Portable metabolomics;

Flowchart for the detection of ABL1-catalysed phosphorylation; DP voltammograms after the phosphorylation reaction and the control experiment.Display OmittedAbelson tyrosine-protein kinase 1 (ABL1) catalysed phosphorylation involves the addition of a phosphate group from ATP to the tyrosine residue on the substrate abltide. The phosphorylation reactions were carried out by incubating ABL1, ATP and the substrate abltide. Adsorption at the glassy carbon electrode surface in either reaction mixtures or control solutions, followed by differential pulse voltammetry in buffer allowed detection of the variation of abltide tyrosine residue oxidation peak reflecting the occurrence of the phosphorylation reaction. The effect of abltide, ATP and ABL1 concentrations as well as the time course of the phosphorylation reaction were studied. The influence of co-adsorption of ABL1, ATP and phosphorylated abltide was evaluated and the conditions for the electrochemical detection of ABL1-catalysed phosphorylation optimised. The Michaelis–Menten constant for abltide binding KM  ∼ 4.5 μM, turnover number k cat  ∼ 11 s−1 and enzyme efficiency k cat/K M  ∼ 2.3 s−1  μM−1 were calculated. The inhibition of ABL1 by imatinib mesylate and danusertib was also electrochemically investigated and IC50 values of 0.53 and 0.08 μM determined.
Keywords: Abelson tyrosine-protein kinase 1; Enzyme kinetics; Inhibition; Electrochemistry; Glassy carbon electrode;

Display OmittedBeing awfully harmful to the environment and human health, the qualitative and quantitative determinations of polycyclic aromatic amines (PAAs) are of great significance. In this paper, a novel core–shell heterostructure of multiwalled carbon nanotubes (MWCNTs) as the core and graphene oxide nanoribbons (GONRs) as the shell (MWCNTs@GONRs) was produced from longitudinal partially unzipping of MWCNTs side walls using a simple wet chemical strategy and applied for electrochemical determination of three kinds of PAAs (1-aminopyrene (1-AP), 1-aminonaphthalene and 3,3′-diaminobiphenyl). Scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, thermogravimetric analysis and electrochemical methods were used to characterize the as-prepared MWCNTs@GONRs. Due to the synergistic effects from MWCNTs and GONRs, the oxidation currents of PAAs at the MWCNTs@GONRs modified glassy carbon (GC) electrode are much higher than that at the MWCNTs/GC, graphene/GC and bare GC electrodes. 1-AP was used as the representative analyte to demonstrate the sensing performance of the MWCNTs@GONRs/GC electrode, and the proposed modified electrode has a linear response range of 8.0–500.0 nM with a detection limit of 1.5 nM towards 1-AP.
Keywords: Polycyclic aromatic amines; Graphene nanoribbons; Carbon nanotubes; Electrochemical sensor; Environmental analysis;

Fabricating a reversible and regenerable electrochemical biosensor for quantitative detection of antibody by using “triplex-stem” DNA molecular switch.Display OmittedA reversible and regenerable electrochemical biosensor is fabricated for quantitative detection of antibody based on “triplex-stem” molecular switches. A hairpin-shaped oligonucleotide (hairpin DNA) labeled with ferrocene (Fc) at the 3′-end is fixed on the gold electrode serving as a signal transduction probe. Its hairpin structure leads Fc close to the surface of gold electrode and produces a strong current signal (on-state). A single-strand oligonucleotide modified with two digoxin molecules on the two arm segments (capture DNA) interact with hairpin DNA with the help of Ag+ ions. The “triplex-stem” DNA forms, which separates Fc from the electrode and reduces the electrochemical signal (off-state). Binding of digoxin antibody to digoxin releases capture DNA from the hairpin DNA, creating an effective “off-on” current signal switch. The stability of the “triplex-stem” structure of hairpin/capture DNA is critical to the signal switch and the sensitivity of the method, which can be adjusted conveniently and efficiently by changing Ag+ concentrations. Based on the “off-on” current signal switch, this biosensor is used to detect digoxin antibody sensitively in blood serum. The linear range is 1.0–500 pg with a correlation coefficient of 0.996, and the detection limit is 0.4 pg. Also, this biosensor shows excellent reversibility and reproducibility, which are significant requirements for practical biosensor applications.
Keywords: Electrochemical biosensor; Antibody detection; Molecular switch; DNA; Digoxin; Silver ion;

Display OmittedIn this publication, a novel solid phase microextraction (SPME) coating functionalized with a DNA aptamer for selective enrichment of a low abundance protein from diluted human plasma is described. This approach is based on the covalent immobilization of an aptamer ligand on electrospun microfibers made with the hydrophilic polymer poly(acrylonitrile-co-maleic acid) (PANCMA) on stainless steel rods. A plasma protein, human α-thrombin, was employed as a model protein for selective extraction by the developed Apt-SPME probe, and the detection was carried out with liquid chromatography/tandem mass spectrometry (LC–MS/MS). The SPME probe exhibited highly selective capture, good binding capacity, high stability and good repeatability for the extraction of thrombin. The protein selective probe was employed for direct extraction of thrombin from 20-fold diluted human plasma samples without any other purification. The Apt-SPME method coupled with LC–MS/MS provided a good linear dynamic range of 0.5–50 nM in diluted human plasma with a good correlation coefficient (R 2  = 0.9923), and the detection limit of the proposed method was found to be 0.30 nM. Finally, the Apt-SPME coupled with LC–MS/MS method was successfully utilized for the determination of thrombin in clinical human plasma samples. One shortcoming of the method is its reduced efficiency in undiluted human plasma compared to the standard solution. Nevertheless, this new aptamer affinity-based SPME probe opens up the possibility of selective enrichment of a given targeted protein from complex sample either in vivo or ex vivo.
Keywords: Aptamer based coating; Electrospun microfiber; Mass spectrometry; Protein quantification; Solid phase microextraction; Thrombin;

Targeted metabolomics in cultured cells and tissues by mass spectrometry: Method development and validation by Anas M. Abdel Rahman; Judy Pawling; Michael Ryczko; Amy A. Caudy; James W. Dennis (53-61).
Display OmittedMetabolomics is the identification and quantitation of small bio-molecules (metabolites) in biological samples under various environmental and genetic conditions. Mass spectrometry provides the unique opportunity for targeted identification and quantification of known metabolites by selective reaction monitoring (SRM). However, reproducibility of this approach depends on careful consideration of sample preparation, chemical classes, and stability of metabolites to be evaluated. Herein, we introduce and validate a targeted metabolite profiling workflow for cultured cells and tissues by liquid chromatography–triple quadrupole tandem mass spectrometry. The method requires a one-step extraction of water-soluble metabolites and targeted analysis of central metabolites that include glycolysis, amino acids, nucleotides, citric acid cycle, and the hexosamine biosynthetic pathway. The sensitivity, reproducibility and molecular stability of each targeted metabolite were assessed under experimental conditions. Quantitation of metabolites by peak area ratio was linear with a dilution over a 4 fold dynamic range with minimal deviation R 2  = 0.98. Inter- and intra-day precision with cells and tissues had an average coefficient of variation <15% for cultured cell lines, and somewhat higher for mouse liver tissues. The method applied in triplicate measurements readily distinguished immortalized cells from malignant cells, as well as mouse littermates based on their hepatic metabolic profiles.
Keywords: Metabolism; Mass spectrometry; Sensitivity; Precision; Diagnostics;

Screening the life cycle of Schistosoma mansoni using high-resolution mass spectrometry by Mônica Siqueira Ferreira; Diogo Noin de Oliveira; Rosimeire Nunes de Oliveira; Silmara Marques Allegretti; Rodrigo Ramos Catharino (62-69).
Display OmittedSchistosomiasis is a common tropical disease caused by Schistosoma species Schistosomiasis' pathogenesis is known to vary according to the worms' strain. Moreover, high parasitical virulence is directly related to eggs release and granulomatous inflammation in the host's organs. This virulence might be influenced by different classes of molecules, such as lipids. Therefore, better understanding of the metabolic profile of these organisms is necessary, especially for an increased potential of unraveling strain virulence mechanisms and resistance to existing treatments. In this report, direct-infusion electrospray high-resolution mass spectrometry (ESI(+)-HRMS) along with the lipidomic platform were employed to rapidly characterize and differentiate two Brazilian S. mansoni strains (BH and SE) in three stages of their life cycle: eggs, miracidia and cercariae, with samples from experimental animals (Swiss/SPF mice). Furthermore, urine samples of the infected and uninfected mice were analyzed to assess the possibility of direct diagnosis. All samples were differentiated using multivariate data analysis, PCA, which helped electing markers from distinct lipid classes; phospholipids, diacylglycerols and triacylglycerols, for example, clearly presented different intensities in some stages and strains, as well as in urine samples. This indicates that biochemical characterization of S. mansoni may help narrowing-down the investigation of new therapeutic targets according to strain composition and aggressiveness of disease. Interestingly, lipid profile of infected mice urine varies when compared to control samples, indicating that direct diagnosis of schistosomiasis from urine may be feasible.
Keywords: Schistosoma mansoni; HR-FTMS; Lipid profile; Life cycle;

Rapid identification of traditional Chinese herbal medicine by direct analysis in real time (DART) mass spectrometry by Yang Wang; Chunmei Li; Liang Huang; Li Liu; Yunlong Guo; Li Ma; Shuying Liu (70-76).
Display OmittedDirect analysis in real time-mass spectrometry (DART-MS) was employed as a novel fast method to identify traditional Chinese herbal medicine (TCHM). In order to obtain high quality mass spectra, the ionization temperature was optimized for every kind of sample. With minimal or no sample pretreatment, major TCHM components, including alkaloids, flavonoids and some ginsenosides, were directly detected within several seconds, while thirteen ginsenosides need derivatization to get good mass spectra. Pseudoginsenoside F11, compound K, protopanaxatriol (PPT) and protopanaxadiol (PPD), for the first time were detected without derivatization. Among five of eight tested Chinese herbal medicines, Rhizoma Corydalis, Bulbus Fritillariae Thunbergii, Arecae Semen, Ramulus Uncariae Cum Uncis and Scutellariae Radix, were first time identified by DART-MS. In addition, the ionization mechanisms of major herbal components, alkaloids, flavonoids and ginsenosides, were discussed in detail. Our results demonstrated that DART-MS could provide a rapid, reliable and environmental friendly method for the rapid identification of TCHM, and may be applicable to other plants.
Keywords: Direct analysis in real time (DART); Time-of-flight mass spectrometry; Alkaloids; Flavonoids; Ginsenosides;

Display OmittedA novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng μL−1 each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.
Keywords: Proteolysis; Peptide mass mapping; Mass spectra; Infrared; Miniature incandescent bulb;

In this study, a novel, sensitive electrochemical immunosensor for simultaneous determination of squamous cell carcinoma associated antigen (SCC-Ag) and carcinoembryonic antigen (CEA) based on poly[3-(1,1′-dimethyl-4-piperidine-methylene) thiophene-2,5-diylchloride] (PDPMT-Cl) and functionalized mesoporous ferroferric oxide nanoparticles (Fe3O4 NPs) for the combined diagnosis of cervical cancer was designed.Display OmittedA novel, sensitive electrochemical immunosensor for simultaneous determination of squamous cell carcinoma associated antigen (SCC-Ag) and carcinoembryonic antigen (CEA) for the combined diagnosis of cervical cancer was designed. The amplification strategy for electrochemical immunoassay was based on poly[3-(1,1′-dimethyl-4-piperidine-methylene) thiophene-2,5-diylchloride] (PDPMT-Cl) and functionalized mesoporous ferroferric oxide nanoparticles (Fe3O4 NPs). PDPMT-Cl dispersed in chitosan solution with enhanced electrical conductivity and solubility was used as matrices to immobilize the first antibodies. Different redox probes (thionine (Th) and ferrocenecarboxylic acid (Fca)) functionalized Fe3O4 NPs incubated with two kinds of secondary antibodies to fabricate the labels. Using an electrochemical analysis technique, two well-separated peaks were generated by Th and Fca, making the simultaneous detection of two analytes on the electrode possible. Under optimized conditions, this method showed wide linear ranges of three orders of magnitude with the detection limits of 4 pg mL−1 and 5 pg mL−1, respectively. The disposable immunosensor possessed excellent clinical value in cervical cancer screening as well as convenient point-of-care diagnostics.
Keywords: Cervical cancer; Simultaneous determination; Polythiophene derivative; Functionalized; Magnetism;

Melamine (M) can hybridize with thymine (T) through the hydrogen-bonding recognition. The combination of melamine with the p-DNA (containing 6 T) modified GNPs induces the aggregation of GNPs. The change in the size of GNPs can be distinguished by DLS technology with high sensitivity.Display OmittedThe migration of melamine monomers from food contact materials has aroused particular attention since the 2008 melamine-tainted milk scandal in China. However, the determination of melamine monomer’s migratory quantity (MMMQ) has remained an open question because of the complex sample pretreatment and the low sensitivity. Based on the hydrogen bonding interaction between DNA thymine and melamine, this paper described a simple and rapid method focusing on the measurement of MMMQ from melamine tableware by gold nanoparticles (GNPs) and dynamic light scattering (DLS). With the presence of probe DNA (p-DNA), the GNPs were stable in NaCl solution (0.06 M), whereas they became aggregated when the p-DNA hybridized with melamine. The change in the hydrodynamic diameter of GNPs could be detected by DLS technology. Under the optimal conditions, the average diameter increased linearly with the concentration of melamine over the range from 5.0 to 320.0 μg L−1, and showed a detection limit of 2.0 μg  L−1 (3σ/slope). The MMMQ was investigated within a range from 6.00 × 10−4 to 2.58 × 10−1  mg dm−2 (n  ≥ 3) in four different food simulants at different temperatures and time points. The results suggest that the DLS method has great potential in the analysis of the migration of melamine monomers.
Keywords: Migration of melamine monomers; Oligonucleotides; Gold nanoparticles; Dynamic light scattering; Melamine tableware;

Display OmittedOrganophosphate triesters are common flame retardants used in a wide variety of consumer products from which they can migrate and pollute the indoor environment. Humans may thus be continuously exposed to several organophosphate triesters which might be a risk for human health. An analytical method based on direct injection of 5 μL urine into an ultra performance liquid chromatography system coupled to a time-of-flight mass spectrometry has been developed and validated to monitor exposure to organophosphate triesters through their respective dialkyl and diaryl phosphate metabolites (DAPs). The targeted analytes were: di-n-butyl phosphate (DNBP), diphenyl phosphate (DPHP), bis(2-butoxyethyl) phosphate (BBOEP), bis(2-chloroethyl) phosphate (BCEP), bis(1-chloro-2-propyl) phosphate (BCPP) and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP). Separation was achieved in less than 3 min on a short column with narrow diameter and small particle size (50 mm × 2.1 mm × 1.7 μm). Different mobile phases were explored to obtain optimal sensitivity. Acetonitrile/water buffered with 5 mM of ammonium hydroxide/ammonium formate (pH 9.2) was the preferred mobile phase. Quantification of DAPs was performed using deuterated analogues as internal standards in synthetic urine (averaged DAP accuracy was 101%; RSD 3%). Low method limits of quantification (MLQ) were obtained for DNBP (0.40 ng mL−1), DPHP (0.10 ng mL−1), BDCIPP (0.40 ng mL−1) and BBOEP (0.60 ng mL−1), but not for the most polar DAPs, BCEP (∼12 ng mL−1) and BCPP (∼25 ng mL−1). The feasibility of the method was tested on 84 morning urine samples from 42 mother and child pairs. Only DPHP was found above the MLQ in the urine samples with geometric mean (GM) concentrations of 1.1 ng mL−1 and 0.57 ng mL−1 for mothers and children respectively. BDCIPP was however, detected above the method limit of detection (MLD) with GM of 0.13 ng mL−1 and 0.20 ng mL−1. While occasionally detected, the GM of DNBP and BBOEP were below MLD in both groups.
Keywords: Human biomonitoring; Urinary metabolites; Time-of-flight mass spectrometry; Flame retardants;