Analytica Chimica Acta (v.795, #C)

Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs) by Vinay G. Joshi; Kantaraja Chindera; Arvind Kumar Singh; Aditya P. Sahoo; Vikas D. Dighe; Dimpal Thakuria; Ashok K. Tiwari; Satish Kumar (1-7).
A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5–10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R 2  = 0.990, which was comparable to the value of R 2  = 0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.
Keywords: Label-free assay; Viral RNA quantification; Visual RNA detection; Gold nanoparticles (AuNPs); Peptide nucleic acid (PNA);

Polyaniline-iron oxide magnetic nanohybrid was synthesized and characterized using various spectroscopic, microstructural and electrochemical techniques. The smart integration of Fe3O4 nanoparticles within the polyaniline (PANI) matrix yielded a mesoporous nanohybrid (Fe3O4@PANI) with high surface area (94 m2  g−1) and average pore width of 12.8 nm. Catechol is quasi-reversibly oxidized to o-quinone and reduced at the Fe3O4@PANI modified electrodes. The amperometric current response toward catechol was evaluated using the nanohybrid and the sensitivity and detection limit were found to be 312 μA μL−1 and 0.2 nM, respectively. The results from electrochemical impedance spectroscopy (EIS) indicated that the increased solution resistance (R s) was due to elevated adsorption of catechol on the modified electrodes. Photoluminescence spectra showed ligand-to-metal charge transfer (LMCT) between p-π orbitals of the phenolate oxygen in catechol and the d-σ* metal orbital of Fe3O4@PANI nanohybrid. Potential dependent spectroelectrochemical behavior of Fe3O4@PANI nanohybrid toward catechol was studied using UV/vis/NIR spectroscopy. The binding activity of the biomagnetic particles to catechol through Brownian relaxation was evident from AC susceptibility measurements. The proposed sensor was used for successful recovery of catechol in tap water samples.
Keywords: PANI-iron oxide nanohybrid; Spectroelectrochemistry; Impedance; Biomagnetic; Catechol;

Display OmittedThe O-methylation of active flavonoids can enhance their antiallergic, anticancerous, and cardioprotective effects depending on the methylation position. Thus, it is biologically and pharmacologically important to differentiate methylated flavonoid regioisomers. In this study, we examined the regioisomers of methylated kaempferol and quercetin using ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. The methyl groups on the flavonoids can generally be cleaved as methyl radicals in a position-independent manner. We found that methyl groups can be cleaved as methane. If there are protons adjacent the methoxy on the flavonol rings, intra-molecule proton transfer can occur via collision-induced dissociation, and one molecule of methane can then be eliminated. The remaining charged fragment ([M+H−CH4]+) reflects the adjacent structure and is specific to the methoxy position. Furthermore, the retro Diels–Alder (RDA) fragmentation of methylated flavonols can generate fragments with the methoxy at the original methylated ring. Combining the position-specific [M+H−CH4]+ fragment with the RDA fragments provides a diagnostic pattern for rapidly identifying methylated regioisomeric flavonols. Along with their retention behaviour, we have successfully identified ten regioisomers of methylated kaempferol and quercetin, which include six compounds previously reported in plants and shown to be biologically active. The developed approach is sensitive, rapid, reliable, and requires few standard compounds. It is highly efficient for characterising the specificity of novel flavonoid O-methyltransferases and can help direct enzymatic or chemical syntheses during the early stages of drug discovery. This method also has potential for use in identifying other methylated isomeric flavonoids.
Keywords: Methylated kaempferol; Methylated quercetin; Regioisomers; Identification; Tandem mass spectrometry fragmentation;

Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed.
Keywords: Quantitative proteome analysis; Isotope labeling; LC–MS; Proteomics; E. coli;

Secondary electrospray ionization-ion mobility-time of flight mass spectrometry (SESI-IM-TOFMS) was used to evaluate common household products and food ingredients for any mass or mobility responses that produced false positives for explosives. These products contained ingredients which shared the same mass and mobility drift time ranges as the analyte ions for common explosives. The results of this study showed that the vast array of compounds in these products can cause either mass or mobility false positive responses. This work also found that two ingredients caused either enhanced or reduced ionization of the target analytes. Another result showed that an IMS can provide real-time separation of ion species that impede accurate mass identifications due to overlapping isotope peak patterns. The final result of this study showed that, when mass and mobility values were used to identify an ion, no false responses were found for the target explosives. The wider implication of these results is that the possibility exists for even greater occurrences of false responses from complex mixtures found in common products. Neither IMS nor MS alone can provide 100% assurance from false responses. IMS, due to its low cost, ease of operation, rugged reliability, high sensitivity and tunable selectivity, will remain the field method of choice for the near future but, when combined with MS, can also reduce the false positive rate for explosive analyses.
Keywords: Ion mobility spectrometry; Mass spectrometry; Secondary electrospray ionization source; Interferences; Personal care products;

Metabolic study of grapevine leaves infected by downy mildew using negative ion electrospray – Fourier transform ion cyclotron resonance mass spectrometry by Loïc Becker; Anne Poutaraud; Grégory Hamm; Jean-François Muller; Didier Merdinoglu; Vincent Carré; Patrick Chaimbault (44-51).
Grapevine is of worldwide economic importance due to wine production. However, this culture is often affected by pathogens causing severe harvest losses. Understanding host–pathogen relationships may be a key to solve this problem. In this paper, we evaluate the direct flow injection by electrospray – Fourier transform ion cyclotron resonance mass spectrometry (MS) of leaf extracts as a rapid method for the study of grapevine response to downy mildew (Plasmopara viticola) attack. The comparison of MS profiles obtained from control and infected leaves of different levels of resistant grapevines highlights several classes of metabolites (mainly saccharides, acyl lipids, hydroxycinnamic acids derivatives and flavonoids) which are identified using high resolution MS and tandem MS (MS/MS). Statistical analyses of 19 markers show a clear segregation between inoculated and healthy samples. This study points out relative high levels of disaccharides, acyl lipids and glycerophosphoinositol in inoculated samples. Sulfoquinovosyl diacylglycerols also emerge as possible metabolites involved in plant defense.
Keywords: High resolution mass spectrometry; Electrospray; Fourier transform ion cyclotron resonance; Metabolic profiling; Grapevine; Downy mildew;

An amperometric biosensor for the determination of l-lysine based on l-lysine-α-oxidase immobilized by co-crosslinking on a platinum electrode previously modified by an overoxidized polypyrrole film is described. The optimization of experimental parameters, such as pH and flow rate, permitted to minimize significantly substrate interferences even using a low specific, commercial enzyme. The relevant biases introduced in the measurement of lysine were just about 1% for l-arginine, l-histidine and l-ornithine, roughly 4% for l-phenylalanine and l-tyrosine. The developed approach allowed linear lysine responses from 0.02 mM up to 2 mM with a sensitivity of 41 nA/(mM × mm2) and a detection limit of 4 μM (S/N = 3). No appreciable loss in lysine sensitivity was observed up to about 40 days. Allowing polypyrrole layer to remove interference from electroactive compounds, the present method revealed suitable to detect l-lysine in a pharmaceutical and cheese sample, showing a good agreement with the expected values.
Keywords: Biosensor; L-lysine determination; L-lysine-α-oxidase; Overoxidized polypyrrole; Flow injection analysis; Food analysis;

We introduce a novel method for encapsulation of curcumin by synthesizing microcapsule containing self-assembled nanoparticles using poly (l-lysine), trisodium citrate and silica sol. Such microcapsules can only be prepared in neutral and alkaline environment and unencapsulated curcumin can be effectively removed by simple centrifugation with encapsulation efficiency 57.34%. The particle sizes are in the range 0.7–3 μm with an effective diameter 1.05 μm. The structure of the microcapsules is dependent upon the solubility of curcumin in the solvent environment, the microcapsule are spherical when prepared in 10% acetone and bowl-shaped/conical when prepared in water suspension, however, the size of these curcumin encapsulated microcapsules remain similar. Fluorescence microscope images confirm that curcumin is predominantly concentrated within the shell wall of the capsules. Photophysical behavior of Micro-curcumin with respect to curcumin alone is evaluated. Release of curcumin is found to be triggered by pH where acidic environment trigger maximum release compared to basic and neutral conditions. Micro-curcumin is as stable as curcumin. Drug release efficiency is found to be 61.44% and the drug release profile of Micro-curcumin follow Higuchi model. It is also revealed that β-diketone group of curcumin responsible for scavenging activity is retained in the Micro-curcumin, thus suggesting applicability of such system as a poorly water soluble drug delivery vehicle. In contrast to other curcumin delivery systems, the presented method is easy, fast and does not need flow rate monitoring device. In addition poly (l-lysine) as a non-toxic and highly stable material that prevents metabolic degradation is used in the present preparation method.
Keywords: Poly-l-lysine; Curcumin; Nanoparticles; Microcapsules; Drug delivery;

A colormetric and fluorescent chemosensor for adenosine-5′-triphosphate based on rhodamine derivative by Chun-Yan Li; Chun-Xiang Zou; Yong-Fei Li; Xue-Fei Kong; Yu Zhou; Yin-Shuang Wu; Wei-Guo Zhu (69-74).
A rhodamine spirolactam derivative (1) was developed as a colormetric and fluorescent chemosensor for adenosine-5′-triphosphate (ATP) via hydrogen bonds interaction. As far as we know, this is the first case to explore ATP-induced ring-opening of spirolactam in rhodamine derivatives. It exhibited a highly sensitive “turn-on” fluorescent response toward ATP with a 47-fold fluorescence intensity enhancement under 20 equiv. of ATP added. The chemosensor can be applied to the quantification of ATP with a linear range covering from 1.0 × 10−7 to 2.0 × 10−4  M and a detection limit of 2.5 × 10−8  M. The experiment results show that the response behavior of 1 toward ATP is pH independent in medium condition (pH 6.0–8.0). Most importantly, the novel chemosensor has well solved the problem of serious interferences from other nucleoside polyphosphates such as ADP and AMP generally met by previously reported typical fluorescent chemosensors for ATP. Moreover, the response of the chemosensor toward ATP is fast (response time less than 3 min). In addition, the chemosensor can be used for the fluorescence assay for protein kinase activity with satisfactory results. The chemosensor for ATP based on hydrogen bonds interaction provided a novel strategy for the design of colormetric and ratiometric fluorescent probes for other target anions with high sensitivity and selectivity.
Keywords: Fluorescence; Chemosensor; Rhodamine; Adenosine-5′-triphosphate;

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples by Constantinos K. Zacharis; Paraskevas D. Tzanavaras; Theano D. Karakosta; Demetrius G. Themelis (75-81).
In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λ ext/λ em  = 340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L−1 CH3COONH4 (pH = 2.5)/CH3CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25–5.0 μmol L−1 and the LOD was 19 nmol L−1. No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.
Keywords: Glutathione; Post-column derivatization; Hydrophilic interaction chromatography; Wines; o-Phthalaldehyde;

One-pot synthesis of phenylphosphonic acid imprinted polymers for tyrosine phosphopeptides recognition in aqueous phase by Quishui Li; Feng Shen; Xiao Zhang; Yufeng Hu; Qingxi Zhang; Lin Xu; Xueqin Ren (82-87).
The tyrosine phosphorylation of proteins plays a vital role in signal transduction pathways. The highly selective enrichment of tyrosine phosphopeptides remains a significant challenge in this area of research because of the low levels of tyrosine phosphorylation in cells. Herein, we report the development of a novel molecularly imprinted polymer (MIP)-based method for the recognition of tyrosine phosphopeptides in aqueous media using an epitope approach. Phenylphosphonic acid, which has been regarded the “epitope” of phosphotyrosine, was used as a template, and commercially available zinc acrylate was used as a functional monomer to prepare the MIP. The one-pot synthetic process was simple, efficient, and the resulting MIPs were low-cost, robust, and recyclable. The MIP demonstrated significant higher levels of adsorption capacity and selectivity for phenylphosphonic acid than the non-imprinting polymer (NIP) over its structural analog benzoic acid. The MIP was also used as a molecular receptor to recognize tyrosine phosphopeptides in aqueous media, and showed a clear preference for tyrosine phosphopeptides over interfering serine peptides compared to TiO2. These results revealed the feasibility of the use of MIPs to effectively mimic the epitope approach, and provided a promising alternative to the immunoaffinity techniques commonly used for capturing tyrosine phosphopeptides.
Keywords: Epitope approach; Molecularly imprinted polymer; Phenylphosphonic acid; Tyrosine phosphopeptides;

The interconversion of cis and trans isomers of dipeptides containing C-terminal proline was studied by dynamic chromatography on zwitterionic chiral stationary phases at temperatures ranging from −15 °C to +45 °C The cistrans isomers could be separated below 0 °C and above 0–10 °C plateau formation and peak coalescence phenomena occurred, which is characteristic for a dynamic process at the time-scale of partitioning. At and above room temperature, full coalescence was observed, which allowed separations of enantiomers without interference from interconversion effects. Analysis of the dynamic elution profiles of the interconverting peptides allowed the determination of isomerization rate constants and thermodynamic activation parameters (isomerization enthalpy, entropy and activation energy). In accordance with established results, isomerization rates and thermodynamic parameters were found to depend on the nature of the N-terminal amino acid. Isomerization barriers were only slightly lower than values determined with other methods but significant differences in the relative contributions of the activation enthalpy and entropy as well as isomerization rates pointed toward selector-moderated isomerization dynamics.
Keywords: Proline; Peptide; cistrans isomerization; Dynamic liquid chromatography; Chiral stationary phase; Kinetic activation parameters;