Analytical and Bioanalytical Chemistry (v.406, #27)
Ariadne’s thread NMR challenge by Reinhard Meusinger (6757-6761).
Solution to the osmium density challenge by John W. Arblaster (6763-6763).
Advanced food analysis by Michel W. F. Nielen; Jana Hajšlová; Rudolf Krska (6765-6766).
is Principal Scientist at RIKILT in Wageningen, The Netherlands, and holds a special chair on Analytical Chemistry at Wageningen University. He is also Scientific Director of TI-COAST, the Dutch public–private partnership on analytical science and technology. His research covers a range of technology for advanced food analysis, including mass spectrometry, biosensing, and combinations thereof. is Head of the Department of Food Analysis and Nutrition, Institute of Chemical Technology, Prague, Czech Republic. Currently, she is the ICTP team leader in charge of scientific and technological aspects of the seven EC FP7 collaborative projects. J. Hajslova participates in many international research activities and under her supervision close collaboration with many world-renowned institutions, for example WHO, FAO, USDA, and the European Commission’s Joint Research Centre has been established. She is also the Czech Republic delegate to the Cooperation Work Programme: Food, Agriculture and Fisheries, Biotechnology. is Full Professor for (Bio-)Analytics and Organic Trace Analysis and is Head of the Department for Agrobiotechnology, IFA-Tulln, with more than 150 co-workers at the University of Natural Resources and Life Sciences, Vienna (BOKU). He obtained his degree in chemistry at the Vienna University of Technology and is an expert in food and feed analysis by chromatographic, mass spectrometric, and immunoanalytical techniques. From 2009 to 2010 he worked for a year as A/Chief of Health Canada’s Food Research Division in Ottawa. He has received six scientific awards and is (co-)author of more than 200 publications.
Mass spectrometric analysis of pharmaceutical adulterants in products labeled as botanical dietary supplements or herbal remedies: a review by Lukas Vaclavik; Alexander J. Krynitsky; Jeanne I. Rader (6767-6790).
The increased availability and use of botanical dietary supplements and herbal remedies among consumers has been accompanied by an increased frequency of adulteration of these products with synthetic pharmaceuticals. Unscrupulous producers may add drugs and analogues of various classes, such as phosphodiesterase type 5 (PDE-5) inhibitors, weight loss, hypoglycemic, antihypertensive and anti-inflammatory agents, or anabolic steroids, to develop or intensify biological effects of dietary supplements or herbal remedies. The presence of such adulterated products in the marketplace is a worldwide problem and their consumption poses health risks to consumers. Analytical methods that allow rapid and reliable testing of dietary supplements for the presence of synthetic drugs are needed to address such fraudulent practices. Mass spectrometry (MS) and hyphenated techniques such as liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) have become primary tools in this endeavor. The present review critically assesses the role and summarizes the applications of MS in the analysis of pharmaceutical adulterants in botanical dietary supplements and herbal remedies. The uses of MS techniques in detection, confirmation, and quantification of known pharmaceutical adulterants as well as in screening for and structure elucidation of unexpected adulterants and novel designer drugs are discussed.
Keywords: Botanical dietary supplements; Herbal remedies; Adulteration; Pharmaceuticals; Mass spectrometry; LC–MS; GC–MS; Ambient ionization methods
Metabolic fingerprinting based on high-resolution tandem mass spectrometry: a reliable tool for wine authentication? by Josep Rubert; Ondrej Lacina; Carsten Fauhl-Hassek; Jana Hajslova (6791-6803).
works in the department of Food Analysis and Nutrition, Institute of Chemical Technology Prague, Czech Republic, as an assistant professor/postdoctoral researcher. His doctoral research focused on the development and optimization of analytical methods using liquid chromatography–mass spectrometry for mycotoxin analysis. Currently, his research mainly involves metabolomics and food authentication based on liquid chromatography and ambient ionization coupled with high-resolution mass spectrometry. works in the Department of Food Analysis and Nutrition, Institute of Chemical Technology Prague, Czech Republic, as a research scientist. His research is mainly focused on the development of liquid chromatography–mass spectrometry-based methods for food and environment analysis as well as on food adulteration and its origin. is a food chemist and works in the Department for Safety in the Food Chain at the Federal Institute for Risk Assessment in Germany. He is Head of the Senior Expert Office for the Import Control of Wine, and has special expertise in wine analysis and appreciation, method development and validation (ring trials), authentication of food and feed, and NMR and stable-isotope analysis. is Head of the Department of Food Analysis and Nutrition at the Institute of Chemical Technology Prague, Czech Republic. Currently, she is the Institute of Chemical Technology Prague team leader in charge of the scientific and technological aspects of seven European Commission FP7 collaborative projects. She participates in many international research activities, and under her supervision there is close collaboration with many world-renowned institutions, such as WHO, FAO, and USDA, and the European Commission’s Joint Research Centre was established. She is also the Czech Republic delegate to the Cooperation Work Programme: Food, Agriculture and Fisheries, Biotechnology. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (MS) and an alternative technology represented by direct analysis in real time coupled with quadrupole time-of-flight MS were investigated for metabolic fingerprinting of 343 red and white wine samples. Direct injection of pure wine and an extraction procedure optimized for isolation of polyphenols were used to compare different analytical and data handling strategies. After data processing and data pretreatment, principal component analysis was initially used to explore the data structure. Initially, the unsupervised models revealed a notable clustering according to the grape varieties, and therefore supervised orthogonal partial least squares discriminant analysis models were created and validated for separation of red and white wines according to the grape variety. The validated orthogonal partial least squares discriminant analysis models based on data (ions) recorded in positive ionization mode were able to classify correctly 95 % of samples. In parallel, authentication parameters, such as origin and vintage, were evaluated, and they are discussed. A tentative identification of markers was performed using accurate mass measurement of MS and MS/MS spectra, different software packages and different online libraries. In this way, different flavonol glucosides and polyphenols were identified as wine markers according to the grape varieties.
Keywords: Wine; Polyphenols; Metabolic fingerprinting; Ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry; Direct analysis in real time–quadrupole time-of-flight mass spectrometry; Chemometrics
Macroscopic and microscopic spatially-resolved analysis of food contaminants and constituents using laser-ablation electrospray ionization mass spectrometry imaging by Michel W. F. Nielen; Teris A. van Beek (6805-6815).
is principal scientist at RIKILT in Wageningen, The Netherlands, and holds a special chair on Analytical Chemistry at Wageningen University. He is also scientific director of TI-COAST, the Dutch public-private partnership on analytical science and technology. His research covers a range of techniques for advanced food analysis, including mass spectrometry, biosensing, and combinations thereof. is Associate Professor in Natural Products Chemistry at the Laboratory of Organic Chemistry of Wageningen University, Wageningen, The Netherlands. As such he has worked on the isolation, purification, identification, and analysis of bioactive or economically relevant metabolites from plants, microbes, animals, or man, for example ginkgolides, antioxidants, paclitaxel, pheromones, natural dyes, and proteins. Laser-ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI) does not require very flat surfaces, high-precision sample preparation, or the addition of matrix. Because of these features, LAESI-MSI may be the method of choice for spatially-resolved food analysis. In this work, LAESI time-of-flight MSI was investigated for macroscopic and microscopic imaging of pesticides, mycotoxins, and plant metabolites on rose leaves, orange and lemon fruit, ergot bodies, cherry tomatoes, and maize kernels. Accurate mass ion-map data were acquired at sampling locations with an x–y center-to-center distance of 0.2–1.0 mm and were superimposed onto co-registered optical images. The spatially-resolved ion maps of pesticides on rose leaves suggest co-application of registered and banned pesticides. Ion maps of the fungicide imazalil reveal that this compound is only localized on the peel of citrus fruit. However, according to three-dimensional LAESI-MSI the penetration depth of imazalil into the peel has significant local variation. Ion maps of different plant alkaloids on ergot bodies from rye reveal co-localization in accordance with expectations. The feasibility of using untargeted MSI for food analysis was revealed by ion maps of plant metabolites in cherry tomatoes and maize-kernel slices. For tomatoes, traveling-wave ion mobility (TWIM) was used to discriminate between different lycoperoside glycoalkaloid isomers; for maize quadrupole time-of-flight tandem mass spectrometry (MS–MS) was successfully used to elucidate the structure of a localized unknown. It is envisaged that LAESI-MSI will contribute to future research in food science, agriforensics, and plant metabolomics. Figure ᅟ
Keywords: Mass spectrometry; Imaging; LAESI; Laser ablation; Food analysis; Agriforensics; Mycotoxins; Pesticides; Glycoalkaloids
Rapid detection of pesticides not amenable to multi-residue methods by flow injection–tandem mass spectrometry by Hans G. J. Mol; Ruud C. J. van Dam (6817-6825).
Flow injection combined with tandem mass spectrometry (MS/MS) was investigated for the rapid detection of highly polar pesticides that are not amenable to multi-residue methods because they do not partition into organic solvents and require dedicated chromatographic conditions. The pesticides included in this study were amitrole, chlormequat, cyromazine, daminozide, diquat, ethephon, fosetyl-Al, glufosinate, glyphosate and its metabolite aminomethylphosphonic acid, maleic hydrazide, mepiquat and paraquat. The composition of the flow-injection solvent was optimized to achieve maximum MS/MS sensitivity. Instrumental limits of detection varied between <0.05 and 1 pg. Fruit, vegetable, cereal, milk and kidney samples were extracted with water (1 % formic acid in case of paraquat/diquat) and ten times diluted in either methanol/0.1 % formic acid, methanol/0.1 % ammonia or acetonitrile/0.1 % ammonia, depending on the pesticide. The ion suppression observed depended strongly on both the matrix and the pesticide. This could be largely compensated for by matrix-matched calibration, but more accurate quantification was obtained by using isotopically labelled standards (commercially available for most of the pesticides studied). The method detection limits ranged from 0.02 mg/kg for chlormequat and mepiquat to 2 mg/kg for maleic hydrazide and were 0.05–0.2 mg/kg for most other pesticide/matrix combinations. This was sufficiently low to test compliance with EU maximum residue limits for many relevant pesticide/commodity combinations. The method substantially reduces the liquid chromatography–MS/MS capacity demand which for many laboratories is prohibitive for inclusion of these pesticides in their monitoring and surveillance programmes. Figure ᅟ
Keywords: Polar pesticides; Flow-injection analysis; Tandem mass spectrometry; Screening; Food
Detection of the food allergen celery via loop-mediated isothermal amplification technique by Celine Zahradnik; Roland Martzy; Robert L. Mach; Rudolf Krska; Andreas H. Farnleitner; Kurt Brunner (6827-6833).
Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0 % for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.
Keywords: Loop-mediated isothermal amplification; Celery; Food allergen; Isothermal amplification; Visual detection
Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach by Celine Zahradnik; Claudia Kolm; Roland Martzy; Robert L. Mach; Rudolf Krska; Andreas H. Farnleitner; Kurt Brunner (6835-6842).
In 2003 the European Commission introduced a 0.9 % threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5 %. A false-negative rate of only 5 % for 1 % GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.
Keywords: 35S promoter; Genetically modified; Transgenic; Isothermal amplification; Screening; Maize; Loop-mediated isothermal amplification; Helicase-dependent amplification
Screening and quantification of pesticide residues in fruits and vegetables making use of gas chromatography–quadrupole time-of-flight mass spectrometry with atmospheric pressure chemical ionization by M. I. Cervera; T. Portolés; F. J. López; J. Beltrán; F. Hernández (6843-6855).
An atmospheric pressure chemical ionization source has been used to enhance the potential of gas chromatography coupled with quadrupole time-of-flight (QTOF) mass spectrometry (MS) for screening and quantification purposes in pesticide residue analysis. A screening method developed in our laboratory for around 130 pesticides has been applied to fruit and vegetable samples, including strawberries, oranges, apples, carrots, lettuces, courgettes, red peppers, and tomatoes. Samples were analyzed together with quality control samples (at 0.05 mg/kg) for each matrix and for matrix-matched calibration standards. The screening strategy consisted in first rapid searching and detection, and then a refined identification step using the QTOF capabilities (MSE and accurate mass). Identification was based on the presence of one characteristic m/z ion (Q) obtained with the low collision energy function and at least one fragment ion (q) obtained with the high collision energy function, both with mass errors of less than 5 ppm, and an ion intensity ratio (q/Q) within the tolerances permitted. Following this strategy, 15 of 130 pesticides were identified in the samples. Afterwards, the quantitation capabilities were tested by performing a quantitative validation for those pesticides detected in the samples. To this aim, five matrices were selected (orange, apple, tomato, lettuce, and carrot) and spiked at two concentrations (0.01 and 0.1 mg/kg), and quantification was done using matrix-matched calibration standards (relative responses versus triphenyl phosphate used as an internal standard). Acceptable average recoveries and relative standard deviations were obtained for many but not all pesticide–matrix combinations. These figures allowed us to perform a retrospective quantification of positives found in the screening without the need for additional analysis. Taking advantage of the accurate-mass full-spectrum data provided by QTOF MS, we searched for a higher number of compounds (up to 416 pesticides) in a second stage by performing extra data processing without any new sample injection. Several more pesticides were detected, confirmed, and/or tentatively identified when the reference standard was unavailable, illustrating in this way the potential of gas chromatography–QTOF MS to detect pesticides in addition to the ones targeted in quantitative analysis of pesticides in food matrices. Figure ᅟ
Keywords: Fruits and vegetables; Pesticides; QuEChERS; Gas chromatography; Quadrupole time-of-flight mass spectrometry; Screening; Quantitative validation
Cellphone-based detection platform for rbST biomarker analysis in milk extracts using a microsphere fluorescence immunoassay by Susann K. J. Ludwig; Hongying Zhu; Stephen Phillips; Ashutosh Shiledar; Steve Feng; Derek Tseng; Leendert A. van Ginkel; Michel W. F. Nielen; Aydogan Ozcan (6857-6866).
Current contaminant and residue monitoring throughout the food chain is based on sampling, transport, administration, and analysis in specialized control laboratories. This is a highly inefficient and costly process since typically more than 99 % of the samples are found to be compliant. On-site simplified prescreening may provide a scenario in which only samples that are suspect are transported and further processed. Such a prescreening can be performed using a small attachment on a cellphone. To this end, a cellphone-based imaging platform for a microsphere fluorescence immunoassay that detects the presence of anti-recombinant bovine somatotropin (rbST) antibodies in milk extracts was developed. RbST administration to cows increases their milk production, but is illegal in the EU and a public health concern in the USA. The cellphone monitors the presence of anti-rbST antibodies (rbST biomarker), which are endogenously produced upon administration of rbST and excreted in milk. The rbST biomarker present in milk extracts was captured by rbST covalently coupled to paramagnetic microspheres and labeled by quantum dot (QD)-coupled detection antibodies. The emitted fluorescence light from these captured QDs was then imaged using the cellphone camera. Additionally, a dark-field image was taken in which all microspheres present were visible. The fluorescence and dark-field microimages were analyzed using a custom-developed Android application running on the same cellphone. With this setup, the microsphere fluorescence immunoassay and cellphone-based detection were successfully applied to milk sample extracts from rbST-treated and untreated cows. An 80 % true-positive rate and 95 % true-negative rate were achieved using this setup. Next, the cellphone-based detection platform was benchmarked against a newly developed planar imaging array alternative and found to be equally performing versus the much more sophisticated alternative. Using cellphone-based on-site analysis in future residue monitoring can limit the number of samples for laboratory analysis already at an early stage. Therewith, the entire monitoring process can become much more efficient and economical. Figure Cellphone-based detection platform for rbST biomarker analysis in milk extracts using a microsphere fluorescence immunoassay
Keywords: Cellphone; Microsphere immunoassay; Anti-rbST antibodies; Biomarker; Milk; Fluorescence imaging
Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish by Katrina Campbell; Sara E. McNamee; Anne-Catherine Huet; Philippe Delahaut; Natalia Vilarino; Luis M. Botana; Mark Poli; Christopher T. Elliott (6867-6881).
Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. ‘an artificial mouse’. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC20) of 4,000, 36, 144 and 46 μg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80 % for all toxins. For validation of the method at the 95 % confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 μg/kg, and the detection capability (CCβ) as a screening method is ≤10 mg/kg, ≤160 μg/kg and ≤400 μg/kg for domoic acid, okadaic acid and saxitoxin, respectively.
Keywords: Multiplex; Phycotoxins; Optical biosensor; Shellfish; Antibody; Validation
Graphene in analytical science by Martin Pumera; Ronen Polsky; Craig Banks (6883-6884).
has been a tenured faculty member at Nanyang Technological University (Singapore) since 2010. He received his PhD degree from Charles University (Czech Republic) in 2001. After two postdoctoral stays (in the USA and Spain), he joined the National Institute for Materials Science (Japan) in 2006 for a tenure-track arrangement, and stayed there until spring 2008, when he accepted a tenured position at the National Institute for Materials Science. In 2009, he was a recipient of an ERC-StG award. He has broad interests in nanomaterials and microsystems, in the specific areas of electrochemistry and synthetic chemistry of carbon nanomaterials, nanomotors, nanotoxicity, and energy-storage devices. is a principal member of the technical staff at Sandia National Laboratories (USA) in the Department of Biosensors and Nanomaterials. He received his PhD degree from New Mexico State University (USA) in 2004. This was followed by a postdoctoral fellowship at the Hebrew University of Jerusalem (Isreal) from 2005 to 2006 before he joined Sandia National Laboratories in 2007. His research areas of interest are electrode arrays, novel chemical functionalizations, porous carbons, and three-dimensional graphene. is a Professor at Manchester Metropolitan University and holds a personal chair in electrochemical and nanotechnology. His current research is directed towards the pursuit of studying the fundamental understanding and applications of nanoelectrochemical systems such as graphene, carbon nanotubes, and nanoparticle-derived sensors, developing novel electrochemical sensors via screen-printing and related techniques, and energy storage and generation based on graphene composites.
Fluorescent sensors using DNA-functionalized graphene oxide by Zhenbao Liu; Biwu Liu; Jinsong Ding; Juewen Liu (6885-6902).
In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNA-based biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNA-functionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescence-quenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.
Keywords: Graphene; DNA; Biosensors; Adsorption; Fluorescence
Recent progress in graphene-material-based optical sensors by Xianghua Deng; Hao Tang; Jianhui Jiang (6903-6916).
Graphene material has been widely used for optical sensors owing to its excellent properties, including high-energy transfer efficiency, large surface area, and great biocompatibility. Different analytes such as nucleic acids, proteins, and small molecules can be detected by graphene-material-based optical sensors. This review provides a comprehensive discussion of graphene-material-based optical sensors focusing on detection mechanisms and biosensor designs. Challenges and future perspectives for graphene-material-based optical sensors are also presented.
Keywords: Graphene material; Graphene oxide; Optical sensor; Fluorescence resonance energy transfer
Sensitive and selective DNA probe based on “turn-on” photoluminescence of C-dots@RGO by Chen-I Wang; Wei-Cheng Wu; Arun Prakash Periasamy; Huan-Tsung Chang (6917-6923).
In this study, highly hydrophilic and photoluminescent sheets of reduced graphene oxide decorated with carbon dots (C-dots@RGO), methylene blue (MB), and a probe DNA have been used for the detection of DNA. The photoluminescence of C-dots@RGO is quenched by MB, which is restored in the presence of a target DNA. The combination of the C-dots@RGO, MB, and a DNA probe is selective for perfectly matched DNA over mismatched DNA, mainly because relative to single-stranded DNA, double-stranded DNA intercalates more strongly with MB, but interacts more weakly with RGO. In the presence of a target DNA, MB intercalates with the as-formed double-stranded DNA and is released from the surface of C-dots@RGO, leading to “turn-on” photoluminescence. The practicality of this assay has been validated by the determination of tumor suppressor gene BRCA1, with linearity over the concentration range from 25 to 250 nM and a limit of detection (LOD, at a signal-to-noise ratio of 3) of 14.6 nM. The C-dots@RGO probe provides higher specificity towards target DNA than towards common salts, carbohydrates, amino acids, and proteins found in real samples. Having the advantages of simplicity, cost-effectiveness, selectivity, and sensitivity, the DNA-P/C-dots@RGO–MB probe on microwells has been successfully employed for the detection of DNA, suggesting its potential for multiple analyses of DNA targets when various DNA probes are employed.
Keywords: Biosensor; Carbon dots; DNA analysis; Reduced graphene oxide; Photoluminescent
A turn-on fluorescence-sensing technique for glucose determination based on graphene oxide–DNA interaction by Hao Zhang; Hui Huang; Zihan Lin; Xingguang Su (6925-6932).
Graphene is a two-dimensional carbon nanomaterial one atom thick. Interactions between graphene oxide (GO) and ssDNA containing different numbers of bases have been proved to be remarkably different. In this paper we propose a novel approach for turn-on fluorescence sensing determination of glucose. Hydrogen peroxide (H2O2) is produced by glucose oxidase-catalysed oxidation of glucose. In the presence of ferrous iron (Fe2+) the hydroxyl radical (•OH) is generated from H2O2 by the Fenton reaction. This attacks FAM-labelled long ssDNA causing irreversible cleavage, as a result of the oxidative effect of •OH, producing an FAM-linked DNA fragment. Because of the weak interaction between GO and short FAM-linked DNA fragments, restoration of DNA fluorescence can be achieved by addition of glucose. Due to the excellent fluorescence quenching efficiency of GO and the specific catalysis of glucose oxidase, the sensitivity and selectivity of this method for GO-DNA sensing are extremely high. The linear range is from 0.5 to 10 μmol L−1 and the detection limit for glucose is 0.1 μmol L−1. The method has been successfully used for analysis of glucose in human serum. Figure ᅟ
Keywords: Fluorescence; Luminescence; Optical sensors; Enzymes
Rapid and simple electrochemical detection of morphine on graphene–palladium-hybrid-modified glassy carbon electrode by Nada F. Atta; Hagar K. Hassan; Ahmed Galal (6933-6942).
A hybrid of reduced graphene oxide–palladium (RGO–Pd) nano- to submicron-scale particles was simultaneously chemically prepared using microwave irradiation. The electrochemical investigation of the resulting hybrid was achieved using cyclic voltammetry and differential pulse voltammetry. RGO–Pd had a higher current response than unmodified RGO toward the oxidation of morphine. Several factors that can affect the electrochemical response were studied, including accumulation time and potential, Pd loading, scan rate, and pH of electrolyte. At the optimum conditions, the concentration of morphine was determined using differential pulse voltammetry in a linear range from 0.34 to 12 μmol L−1 and from 14 to 100 μmol L−1, with detection limits of 12.95 nmol L−1 for the first range. The electrode had high sensitivity toward morphine oxidation in the presence of dopamine (DA) and of the interference compounds ascorbic acid (AA) and uric acid (UA). Electrochemical determination of morphine in a spiked urine sample was performed, and a low detection limit was obtained. Validation conditions including reproducibility, sensitivity, and recovery were evaluated successfully in the determination of morphine in diluted human urine.
Keywords: Electrochemical sensor; Reduced graphene oxide; Palladium nanoparticles; Morphine
A novel electrochemical DNA biosensor construction based on layered CuS–graphene composite and Au nanoparticles by Chun-Xuan Xu; Qiu-Ge Zhai; Yu-Jie Liu; Ke-Jing Huang; Lu Lu; Ke-Xin Li (6943-6951).
A novel CuS–graphene (CuS-Gr) composite was synthesized to achieve excellent electrochemical properties for application as a DNA electrochemical biosensor. CuS-Gr composite was prepared by a hydrothermal method, in which two-dimensional graphene served as a two-dimensional conductive skeleton to support CuS nanoparticles. A sensitive electrochemical DNA biosensor was fabricated by immobilizing single-stranded DNA (ss-DNA) labeled at the 5′ end using 6-mercapto-1-hexane (HS-ssDNA) on the surface of Au nanoparticles (AuNPs) to form ssDNA-S–AuNPs/CuS-Gr, and hybridization sensing was done in phosphate buffer. Cyclic voltammetry and electrochemical impedance spectroscopy were performed for the characterization of the modified electrodes. Differential pulse voltammetry was applied to monitor the DNA hybridization using an [Fe(CN)6]3−/4− solution as a probe. Under optimum conditions, the biosensor developed exhibited a good linear relationship between the current and the logarithm of the target DNA concentration ranging from 0.001 to 1 nM, with a low detection limit of 0.1 pM (3σ/S). The biosensor exhibited high selectivity to differentiate one-base-mismatched DNA and three-base-mismatched DNA. The results indicated that the sensing platform based on CuS-Gr provides a stable and conductive interface for electrochemical detection of DNA hybridization, and could easily be extended to the detection of other nucleic acids. Graphical abstracts ᅟ
Keywords: CuS–graphene composite; Gold nanoparticles; DNA biosensor; DNA hybridization
Picomolar detection of mercury (II) using a three-dimensional porous graphene/polypyrrole composite electrode by Meng Wang; Wenjing Yuan; Xiaowen Yu; Gaoquan Shi (6953-6956).
A polypyrrole (PPy)-functionalized three-dimensional (3D) porous electrode of electrochemically reduced graphene oxide (ErGO) has been prepared by electrochemical deposition. This PPy-modified 3D-ErGO electrode was used for the electrochemical detection of Hg2+ ions, and it exhibited high sensitivity and selectivity. Furthermore, the limit of detection (LOD) was measured to be as low as 0.03 nM (30 ppt), and this value is much lower than the guideline value of 2 ppb for drinking water given by the World Health Organization.
Keywords: Polypyrrole; Reduced graphene oxide; Hg2+ ions; Porous electrode
Current trends in single cell analysis by Petra Dittrich; Norbert Jakubowski (6957-6961).
has been Assistant Professor for Bioanalytics at ETH Zurich (Switzerland) since 2008. She studied chemistry at Bielefeld University (Germany) and Universidad de Salamanca (Spain), and earned her PhD at the Max Planck-Institute for Biophysical Chemistry (Göttingen, Germany) in 2003. After another year as a postdoctoral fellow at the MPI Göttingen, she worked at the Institute for Analytical Sciences (Dortmund, Germany) (2004–2008). For research stays, she was at the Cornell University (2002) and the University of Tokyo (2005). Her research focuses on the miniaturization of high-sensitivity devices for chemical and biological analyses, particularly for single-cell analysis, and the development of new methods for microfluidic-aided organization of materials and artificial cells. is presently head of the division “Inorganic Trace Analysis” at the BAM - Federal Institute for Materials Research and Testing, Berlin, Germany (since 2009). He studied plasma-physics at the University of Essen/Duisburg and worked at the Institute for Analytical Sciences (Dortmund, Germany) (1982-2009). He received his PhD at the University of Stuttgart Hohenheim in 1991. His research interests are related to analytical chemistry in general with special interest in the development of instruments, methods, and problem-orientated procedures based on the use of plasma sources (inductively coupled plasma, glow discharge) for elemental mass spectrometry of solid and liquid samples. At BAM he recently started a new research direction towards using laser ablation ICP-MS for bio- and immunoimaging of tissues and single cells.
Trends in single-cell analysis by use of ICP-MS by Larissa Mueller; Heike Traub; Norbert Jakubowski; Daniela Drescher; Vladimir I. Baranov; Janina Kneipp (6963-6977).
The analysis of single cells is a growing research field in many disciplines such as toxicology, medical diagnosis, drug and cancer research or metallomics, and different methods based on microscopic, mass spectrometric, and spectroscopic techniques are under investigation. This review focuses on the most recent trends in which inductively coupled plasma mass spectrometry (ICP-MS) and ICP optical emission spectrometry (ICP-OES) are applied for single-cell analysis using metal atoms being intrinsically present in cells, taken up by cells (e.g., nanoparticles), or which are artificially bound to a cell. For the latter, especially element tagged antibodies are of high interest and are discussed in the review. The application of different sample introduction systems for liquid analysis (pneumatic nebulization, droplet generation) and elemental imaging by laser ablation ICP-MS (LA-ICP-MS) of single cells are highlighted. Because of the high complexity of biological systems and for a better understanding of processes and dynamics of biologically or medically relevant cells, the authors discuss the idea of “multimodal spectroscopies.”
Keywords: Bioanalytical methods; Cell systems/single cell analysis; Mass spectrometry/ICP-MS
Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells by Stéphane Roudeau; Asuncion Carmona; Laura Perrin; Richard Ortega (6979-6991).
X-ray chemical element imaging has the potential to enable fundamental breakthroughs in the understanding of biological systems because chemical element interactions with organelles can be studied at the sub-cellular level. What is the distribution of trace metals in cells? Do some elements accumulate within sub-cellular organelles? What are the chemical species of the elements in these organelles? These are some of the fundamental questions that can be addressed by use of X-ray chemical element imaging with synchrotron radiation beams. For precise location of the distribution of the elements, identification of cellular organelles is required; this can be achieved, after appropriate labelling, by use of fluorescence microscopy. As will be discussed, this approach imposes some limitations on sample preparation. For example, standard immunolabelling procedures strongly modify the distribution of the elements in cells as a result of the chemical fixation and permeabilization steps. Organelle location can, however, be performed, by use of a variety of specific fluorescent dyes or fluorescent proteins, on living cells before cryogenic fixation, enabling preservation of element distribution. This article reviews the methods used for fluorescent organelle labelling and X-ray chemical element imaging and speciation of single cells. Selected cases from our work and from other research groups are presented to illustrate the potential of the combination of the two techniques. Figure Synchrotron X-ray fluorescence distribution maps of Ca, P and S in yeast cells. Elemental distribution maps (green color scale) were combined with the image of vacuoles labeled with Arg-CMAC (red color scale). The yellow signal of superposed images shows that Ca and P are preferentially located within the vacuole.
Keywords: Single cell; Organelle; Synchrotron; X-ray fluorescence; GFP; Metals; Fluorescence microscopy
Label-free in vitro visualization and characterization of caveolar bulbs during stimulated re-epithelialization by Melissa Watkins-Mariani; Tanja Deckert-Gaudig; Volker Deckert (6993-7002).
Tip-enhanced Raman scattering (TERS) was paired with real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to characterize lipid aggregates during stimulated re-epithelialization using an in vitro wound healing model. In this study, lipid fluctuations in the plasma membrane of epidermal keratinocytes were studied at multiple time points post-wounding. TERS measurements for the first time were also combined with sample analysis after initial wounding and 24 h of wound healing. This enabled simultaneous visualization and characterization of caveolar bulb distribution during wound healing stages, providing noninvasive insight into their associated lipid structure and coating protein, caveolin, in the nanometer range. The combination of Raman spectroscopy and scanning probe microscopy in TERS gives access to topographic and chemical structure information in a single experiment. It is the intrinsic specificity and sensitivity of TERS that enable this discrete detection of cell surface components on the nanometer scale. In contrast with competing biochemical methods, the applied technique does not interfere with the cellular composition, enabling lipid structure analysis without digestion or detergents, and displayed great potential for future biological in vivo studies. Figure tip_enhanced Raman spectroscopy of cell caveolar bulbs formed on a wound healing model
Keywords: TERS; Caveolae; Caveolin; Wound healing; Sphingolipid
Relating surface-enhanced Raman scattering signals of cells to gold nanoparticle aggregation as determined by LA-ICP-MS micromapping by Tina Büchner; Daniela Drescher; Heike Traub; Petra Schrade; Sebastian Bachmann; Norbert Jakubowski; Janina Kneipp (7003-7014).
The cellular response to nanoparticle exposure is essential in various contexts, especially in nanotoxicity and nanomedicine. Here, 14-nm gold nanoparticles in 3T3 fibroblast cells are investigated in a series of pulse-chase experiments with a 30-min incubation pulse and chase times ranging from 15 min to 48 h. The gold nanoparticles and their aggregates are quantified inside the cellular ultrastructure by laser ablation inductively coupled plasma mass spectrometry micromapping and evaluated regarding the surface-enhanced Raman scattering (SERS) signals. In this way, both information about their localization at the micrometre scale and their molecular nanoenvironment, respectively, is obtained and can be related. Thus, the nanoparticle pathway from endocytotic uptake, intracellular processing, to cell division can be followed. It is shown that the ability of the intracellular nanoparticles and their accumulations and aggregates to support high SERS signals is neither directly related to nanoparticle amount nor to high local nanoparticle densities. The SERS data indicate that aggregate geometry and interparticle distances in the cell must change in the course of endosomal maturation and play a critical role for a specific gold nanoparticle type in order to act as efficient SERS nanoprobe. This finding is supported by TEM images, showing only a minor portion of aggregates that present small interparticle spacing. The SERS spectra obtained after different chase times show a changing composition and/or structure of the biomolecule corona of the gold nanoparticles as a consequence of endosomal processing.
Keywords: Gold nanoparticles; Surface-enhanced Raman scattering; LA-ICP-MS; Fibroblast; Cell; Particle aggregation; Endosome
Real-time monitoring of immobilized single yeast cells through multifrequency electrical impedance spectroscopy by Zhen Zhu; Olivier Frey; Felix Franke; Niels Haandbæk; Andreas Hierlemann (7015-7025).
We present a microfluidic device, which enables single cells to be reliably trapped and cultivated while simultaneously being monitored by means of multifrequency electrical impedance spectroscopy (EIS) in the frequency range of 10 kHz–10 MHz. Polystyrene beads were employed to characterize the EIS performance inside the microfluidic device. The results demonstrate that EIS yields a low coefficient of variation in measuring the diameters of captured beads (~0.13 %). Budding yeast, Saccharomyces cerevisiae, was afterwards used as model organism. Single yeast cells were immobilized and measured by means of EIS. The bud growth was monitored through EIS at a temporal resolution of 1 min. The size increment of the bud, which is difficult to determine optically within a short time period, can be clearly detected through EIS signals. The impedance measurements also reflect the changes in position or motion of single yeast cells in the trap. By analyzing the multifrequency EIS data, cell motion could be qualitatively discerned from bud growth. The results demonstrate that single-cell EIS can be used to monitor cell growth, while also detecting potential cell motion in real-time and label-free approach, and that EIS constitutes a sensitive tool for dynamic single-cell analysis. Figure ᅟ
Keywords: Microfluidics; Single-cell analysis; Electrical impedance spectroscopy; Cell trapping; S. cerevisiae
Single-cell sphingosine kinase activity measurements in primary leukemia by Alexandra J. Dickinson; Sally A. Hunsucker; Paul M. Armistead; Nancy L. Allbritton (7027-7036).
Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39 ± 26 % per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16 ± 25 %, 11 ± 26 %, and 13 ± 23 % in a chronic myelogenous leukemia (CML) patient, an AML patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87 % of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99 % of the cell population, 19 ± 36 % of the loaded sphingosine was phosphorylated. Thus, the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12 ± 0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer. Figure Phosphorylation of a fluorescent sphingosine kinase reporter was used to measure single-cell SK activity in primary cells from leukemic patients. Peripheral blood mononuclear cells as well as enriched leukemic blasts were analyzed.
Keywords: Single-cell analysis; Sphingosine kinase; Capillary electrophoresis; Leukemia; Primary cells
Complexity of fatty acid distribution inside human macrophages on single cell level using Raman micro-spectroscopy by Clara Stiebing; Christian Matthäus; Christoph Krafft; Andrea-Anneliese Keller; Karina Weber; Stefan Lorkowski; Jürgen Popp (7037-7046).
Macrophages are phagocytic cells which are involved in the non-specific immune defense. Lipid uptake and storage behavior of macrophages also play a key role in the development of atherosclerotic lesions within walls of blood vessels. The allocation of exogenous lipids such as fatty acids in the blood stream dictates the accumulation and quantity of lipids within macrophages. In case of an overexposure, macrophages transform into foam cells because of the large amount of lipid droplets in the cytoplasm. Raman micro-spectroscopy is a powerful tool for studying single cells due to the combination of microscopic imaging with spectral information. With a spatial resolution restricted by the diffraction limit, it is possible to visualize lipid droplets within macrophages. With stable isotopic labeling of fatty acids with deuterium, the uptake and storage of exogenously provided fatty acids can be investigated. In this study, we present the results of time-dependent Raman spectroscopic imaging of single THP-1 macrophages incubated with deuterated arachidonic acid. The polyunsaturated fatty acid plays an important role in the cellular signaling pathway as being the precursor of icosanoids. We show that arachidonic acid is stored in lipid droplets but foam cell formation is less pronounced as with other fatty acids. The storage efficiency in lipid droplets is lower than in cells incubated with deuterated palmitic acid. We validate our results with gas chromatography and gain information on the relative content of arachidonic acid and its metabolites in treated macrophages. These analyses also provide evidence that significant amounts of the intracellular arachidonic acid is elongated to adrenic acid but is not metabolized any further. The co-supplementation of deuterated arachidonic acid and deuterated palmitic acid leads to a non-homogenous storage pattern in lipid droplets within single cells. Figure a ᅟ
Keywords: Macrophages; Lipid droplet; Arachidonic acid; Atherosclerosis; Gas chromatography; Raman spectroscopy