Analytical and Bioanalytical Chemistry (v.410, #1)
ABC Spotlight on paper-based strips analytics by Günter Gauglitz (1-3).
is Senior Professor at the Eberhard Karls University of Tübingen working in analytical and physical chemistry. He was Chairman of the Division of Analytical Chemistry of the German Chemical Society, and chaired the Europt(r)ode VIII meeting. For more than 15 years his main scientific interests have centered on research and development in chemical and biochemical sensors, with special focus on the characterization of interfaces of polymers and biomembrane surfaces, spectroscopic techniques, use of spectral interferometry to monitor changes in the optical thickness of thin layers, and the effects of Fresnel reflectivity at interfaces. He has been an editor of Analytical and Bioanalytical Chemistry since its foundation in 2002.
Analytical evaluation of sensor measurements by Günter Gauglitz (5-13).
is a senior professor at the Eberhard Karls University of Tübingen working in analytical and physical chemistry. He was Chairman of the German Chemical Society (Gesellschaft Deutscher Chemiker) Division of Analytical Chemistry and chaired the Europt(r)ode VIII meeting. For more than 15 years his main scientific interests have centered on research and development in chemical and biochemical sensors, with special focus on the characterization of interfaces of polymers and biomembrane surfaces, spectroscopic techniques, use of spectral interferometry to monitor changes in optical thickness of thin layers, and the effects of Fresnel reflectivity at interfaces. He has been an editor of Analytical and Bioanalytical Chemistry since 2002.
Collision induced dissociation mass spectrometry challenge by Daniel G. Beach; Wojciech Gabryelski (15-17).
Solution to pink tea challenge by Lucia D’Ulivo (19-20).
A new strategy for metal labeling of glycan structures in antibodies by Lena Ruhe; Stefanie Ickert; Sebastian Beck; Michael W. Linscheid (21-25).
Quantitative analysis of complex proteins is a challenging task in modern bioanalytical chemistry. Commonly available isotope labels are still suffering from limitations and drawbacks, whereas new metal labels open numerous possibilities in mass spectrometric analyses. In this work, we have developed a new metal labeling strategy to tag glycan structures of proteins, more particularly antibodies. The oligosaccharide glycans were selectively trimmed to the last N-acetylglucosamine to which an artificial azide containing galactose residue was bound. This azide can be used for subsequent cycloaddition of an alkyne. Therefore, we developed a lanthanide-containing macrocyclic reagent to selectively connect to this azido galactose. In summary, the glycan structures of an antibody can be labeled with a metal functionality using this approach. Furthermore, the functionality of the antibodies can be fully maintained by labeling the Fc glycans instead of using labeling reagents that target amino or thiol groups. This approach enables the possibility of using elemental, besides molecular mass spectrometry, for quantitative analyses or imaging experiments of antibodies in complex biological samples. Graphical abstract Antibody labeling at sugar moieties with rare earth elements to enable application in elemental mass spectrometry.
Keywords: Antibody; Metal labeling; Glycans; DOTA; Lanthanide
Monitoring enzymatic degradation of emerging contaminants using a chip-based robotic nano-ESI-MS tool by Lara F. Stadlmair; Thomas Letzel; Johanna Graßmann (27-32).
Up to now, knowledge of enzymes capable of degrading various contaminants of emerging concern (CEC) is limited, which is especially due to the lack of rapid screening methods. Thus, a miniaturized high-throughput setup using a chip-based robotic nanoelectrospray ionization system coupled to mass spectrometry has been developed to rapidly screen enzymatic reactions with environmentally relevant CECs. Three laccases, two tyrosinases, and two peroxidases were studied for their ability to transform ten pharmaceuticals and benzotriazole. Acetaminophen was most susceptible to enzymatic conversion by horseradish peroxidase (HRP), laccase from Trametes versicolor (LccTV), and a tyrosinase from Agaricus bisporus (TyrAB). Diclofenac and mefenamic acid were converted by HRP and LccTV, whereas sotalol was solely amenable to HRP conversion. Benzotriazole, carbamazepine, gabapentin, metoprolol, primidone, sulfamethoxazole, and venlafaxine remained persistent in this study. The results obtained here emphasize that enzymes are highly selective catalysts and more effort is required in the use of fast monitoring technologies to find suitable enzyme systems. Despite the methodological limitations discussed in detail, the automated tool provides a routine on-line screening of various enzymatic reactions to identify potential enzymes that degrade CECs. Graphical abstract A chip-based robotic nano-ESI-MS tool to rapidly monitor enzymatic degradation of environmentally relevant emerging contaminants
Keywords: Mass spectrometry; Miniaturization; High-throughput screening; Robotic nano-ESI; Oxidative enzymes; Emerging contaminants
Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection by B. Leticia Fernández-Carballo; Christine McBeth; Ian McGuiness; Maxim Kalashnikov; Christoph Baum; Salvador Borrós; Andre Sharon; Alexis F. Sauer-Budge (33-43).
, PhD, finished her doctoral work in March 2017, which was focused on the design of low-cost in vitro diagnostic devices (including nucleic acid assays and immunoassays) for low-resource settings. Her PhD research was conducted at the Institut Químic de Sarrià and the Fraunhofer Center for Manufacturing Innovation. She is currently working at the Foundation for Innovative New Diagnostics in Geneva (Switzerland). , PhD, is a research scientist at the Fraunhofer Center for Manufacturing Innovation at Boston University. Having trained in molecular biophysics and infectious disease, she no conducts in-house research into point-of-care diagnostic development, rapid antibiotic susceptibility testing via microfluidics, and 3D bioprinting of tissues. She also partners closely with the engineering teams at the Fraunhofer Center for Manufacturing Innovation to help top biotechnology companies move their favorite R & D projects to the industrial scale. is a manufacturing engineer at Tesla. He is responsible for developing automation systems for Tesla’s electric motors. He was formerly a project engineer at Fraunhofer USA, where he developed custom solutions for clients, including microfluidic devices, laboratory equipment, and industrial automation. He holds an MS degree in mechanical engineering from Boston University. , PhD, is a research scientist at the Fraunhofer Center for Manufacturing Innovation located at Boston University (Brookline, MA, USA). His research is focused on combining microfluidics and microscopy to develop novel diagnostics methods in the field of infectious diseases. , PhD, is Head of the Precision Machines Department of the Fraunhofer Institute for Production Technology in Aachen, Germany. He is active in the research field of ultraprecision technologies and polymer replication by injection molding and roll-to-roll processes, targeting optics, and microfluidics. , PhD, is Director of the Materials Engineering Group (GEMAT) at the Institut Químic de Sarrià (IQS), Ramon Llull University, Barcelona, and Head of the Chemical Engineering and Materials Science Department in the IQS School of Engineering. He has been working for several years on the development of functional materials (mainly polymers) in different fields, mainly in biomaterials. He has developed bioactive surfaces for biological applications and microfluidics. PhD, is the Director of the Fraunhofer Center for Manufacturing Innovation and Professor of Mechanical Engineering at Boston University. His research and passion lies at the intersection of engineering and life sciences. His work focuses on accelerating the diagnosis of disease, and allowing scientific research and experimentation to proceed at a faster rate by improving the efficiency of sample preparation and process yield through automation. PhD, is a managing biotechnology scientist at Exponent and Adjunct Associate Professor in the Biomedical Engineering Department at Boston University. She was formerly the Senior Research Scientist at the Fraunhofer Center for Manufacturing Innovation leading the development of point-of-care diagnostics, tissue engineering, next-generation medical devices, and scientific instrumentation. One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip
Keywords: Lab on a chip; Quantitative reverse transcription polymerase chain reaction; RNA-based virus detection; Infectious diseases; Point of care; Ebola virus
Monitoring of post-mortem changes of saliva N-glycosylation by nano LC/MS by Bum Jin Kim; Chanyoung Han; Hantae Moon; Joseph Kwon; Ik-Soon Jang; Si-Keun Lim; Ki-Won Park; Jong-Soon Choi; Hyun Joo An (45-56).
is a graduate student at the Graduate School of Analytical Science and Technology (GRAST) at the Chungnam National University in Daejeon, Korea. His research mainly focuses on forensic science using MS-based glycomics approach. is a graduate student at the Graduate School of Analytical Science and Technology (GRAST) in Daejeon, Korea. His research interest is the development of forensic platform for analysis and identification of human body fluids using mass spectrometry-based glycomics. is a principle scientist and the Team Leader of Biological Disaster Research Group at the Korea Basic Science Institute in Daejeon, Korea. His research interest is the integrated profiling of global proteomic, metabolomic, transcriptomic responses to virus for neutralizing. is a principle scientist of the Division of Bio convergence at the Korea Basic Science Institute in Daejeon, Korea. His research interests are signal transduction for aging and screening for new drugs MOA using TF expression network. has been a forensic DNA specialist in the National Forensic Service in South Korea since 1997. His recent research interests involve DNA methylation analysis for body fluids identification and age prediction, PNA-based SNPs analysis, forensic phenotyping for prediction of biogeographical ancestry. is a director general of the Department of Forensic Biology and Chemistry in National Forensic Service in South Korea. His recent research interests involve Forensic Science, Forensic Biology and Forensic Genetics. is the principal researcher in the Korea Basic Science Institute. His main research is oriented in the analytical forensic science using functional proteomics. is a professor at the Graduate School of Analytical Science and Technology at the Chungnam National University in Daejeon, Korea. She is the director of the Asia Glycomics Reference Site (AGRS), which develops and validates new analytical platforms for glycomic and glycoproteomic analysis using cutting-edge facilities and equipment. Her research focuses on bioanalytical mass spectrometry, with applications to glycomics, proteomics, and glycoproteomics. The estimation of post-mortem interval (PMI) is a crucial part for investigations of crime and untimely deaths in forensic science. However, standard methods of PMI estimation are easily confounded by extenuating circumstances and/or environmental factors. Therefore, a panel of PMI markers obtained from a more acceptable and accurate method is necessary to definitely determine time of death. Saliva, one of the vital fluids encountered at crime scenes, contains various glycoproteins that are highly affected by biochemical environment. Here, we investigated saliva N-glycans between live and dead rats to determine the alteration of N-glycans using an animal model system because of the limitation of saliva collection from recently deceased humans. Rat saliva samples were collected both before and after death. N-Glycans were enzymatically released by PNGase F without any glycoprotein extraction. Released native glycans were purified and enriched by PGC-SPE. About 100 N-glycans were identified, profiled, and structurally elucidated by nano LC/MS and tandem MS. Sialylated N-glycans were exclusively present in abundance in live rat saliva whereas non-sialylated N-glycans including LacdiNAc disaccharides were detected in high level following death. Through in-depth investigations using quantitative comparison and statistical analysis, 14 N-glycans that significantly changed after death were identified as the potential marker candidates for PMI estimation. To the best of our knowledge, this is the first study to monitor the post-mortem changes of saliva glycosylation, with obvious forensic applications.
Keywords: Saliva; Glycosylation; Mass spectrometry; Post-mortem
Fast automated online xylanase activity assay using HPAEC-PAD by Christin Cürten; Nico Anders; Niels Juchem; Nina Ihling; Kristina Volkenborn; Andreas Knapp; Karl-Erich Jaeger; Jochen Büchs; Antje C. Spiess (57-69).
is a research assistant at the Chair of Biochemical Engineering at RWTH Aachen University. She identifies and describes the effects of biomass pretreatment and the enzymatic hydrolysis of lignocellulosic biomass mechanistically to optimize the conditions applied in biorefinery processes. heads a junior research group aiming at overcoming analytical bottlenecks in biorefinery processes at RWTH Aachen University. The main research interests of his group are the analysis of complex biomass matrices, simultaneous chromatographic separation of biomass-derived products from cellulose, hemicellulose, pectin, and lignin, as well as the automation of enzyme assays. studied biotechnology at the University of Applied Science Bingen am Rhein. After finishing his bachelor’s degree, he studied molecular and applied biotechnology at RWTH Aachen and worked on the development of a method for automated enzyme analysis during his master’s thesis. works as a research assistant at the Chair of Biochemical Engineering at Aachen University, which is headed by Prof. Dr.-Ing. Jochen Büchs. She characterizes bacterial secretion systems in order to learn how to optimize the heterologous production of different proteins such as biomass-degrading enzymes. holds a Master of Science degree in biology and currently works as a PhD student in the Bacterial Cell Factories group at the Institute of Molecular Enzyme Technology of the Heinrich Heine University Düsseldorf. Her PhD project deals with the expression and secretion of biomass-degrading enzymes by Bacillus subtilis. leads the Bacterial Cell Factories research group at the Institute of Molecular Enzyme Technology of Heinrich Heine University Düsseldorf. His research focuses on studying the production and secretion of biotechnologically relevant proteins and enzymes by bacteria. is Professor and Director of the Institute of Molecular Enzyme Technology of Heinrich Heine University Düsseldorf, Director at the Institute of Bio- and Geosciences IBG-1: Biotechnology at Forschungszentrum Jülich GmbH, and serves as Vice President of the Cluster of Industrial Biotechnology CLIB2021. His main research interests include the identification, production, and characterization of bacterial enzymes as well as determining the structure, function, and biotechnological applications of fluorescent proteins. is Professor and Head of the Chair of Bioprocess Engineering at RWTH Aachen University. The core competence of the chair is the characterization of processes in bioreactors of all scales, from microplates to fermenters. The focus is on the development and application of innovative methods for online process analysis and control. is Professor and Head of the Institute of Biochemical Engineering at TU Braunschweig. Her research mainly focuses on quantitatively elucidating the molecular phenomena in heterogeneous bioprocesses, e.g., in enzymatic hydrolysis of the pretreated biomass. This involves the generation of precise data and the application of mechanistic modeling approaches. In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35–1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation
Keywords: Automatic activity assay; Xylanase; HPAEC-PAD; Enzyme
Enhancing the immunofluorescent sensitivity for detection of Acidovorax citrulli using fluorescein isothiocyanate labeled antigen and antibody by Haijuan Zeng; Duoqiang Zhang; Xuzhao Zhai; Shujuan Wang; Qing Liu (71-77).
A rapid lateral flow immunochromatographic strip (ICS) using fluorescein isothiocyanate (FITC) labeled antigen and antibody was developed for the detection of Acidovorax citrulli (Ac) in melons and vegetable samples. In the ICS, signal amplification was realized based on antigen Ac and anti-Ac monoclonal antibody (McAb) 4F conjugated with FITC, respectively, which were forming two probes. The control line and the test line were obtained by immobilizing the goat anti-mouse IgG antibody and anti-Ac McAb 6D on both sides of the nitrocellulose membrane. The visual detection limit of the strip was 105 CFU/mL, which was 10-fold sensitive compared to the strip of FITC only labeling antigen or antibody. Signal amplification ICS was successfully applied to the detection of Ac in melon and vegetable samples with less detection time and operation procedures compared to the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. This is the first report of using FITC labeled antigen and McAb as dual fluorescent probes to develop a direct-type immunofluorescence strip for the rapid and sensitive detection of Ac, which demonstrates a powerful tool for rapidly screening Ac in plant materials and other samples. Graphical abstract The schematic presentation of the test strip (a) and the positive result (b) or negative result (c) of the test strip
Keywords: FITC; Antigen and antibody dual labeled; Immunoassay; Acidovorax citrulli
Reductive oxyamination: a method for the qualitative and quantitative analysis of monosaccharides with a new aminooxy reagent using high-performance liquid chromatography with fluorescence detection by Manman Ding; Zhaobing Guan; Hongwei Cai; Yiyong Huang; Yawei Lin; Xiaosong Hu (79-89).
Derivatization of carbohydrates with aminooxy agents to form oximes can be used for qualitative and quantitative analysis of carbohydrates; however, the formation of isomeric products limits its application. A new reductive oxyamination procedure developed for the analysis of monosaccharides with a novel fluorescent O-substituted aminooxy reagent, 4-((aminooxy)methyl)-6-chloro-7-hydroxycoumarin (AOCC), is reported. In this procedure, monosaccharides undergo an oxime formation reaction with AOCC and are then readily reduced with 2-picoline–borane, followed by analysis with high-performance liquid chromatography with fluorescence detection. Good separation of five monosaccharide derivatives was achieved within 40 min with acetonitrile–water–tetrahydrofuran as the mobile phase. The detection limits were on the order of femtomoles. The linear range was 0.2–4000 nM, with a good correlation coefficient (R ≥ 0.9985). Furthermore, the method was applied for analysis of real samples, such as bovine milk powder, without complicated and tedious sample treatment. This reductive oxyamination method circumvents the problem caused by oxime isomers and can be used for the highly sensitive and selective analysis of monosaccharides with high accuracy, providing an effective and promising method for the analysis of carbonyls with aminooxy agents.
Keywords: Reductive oxyamination; Precolumn fluorescent derivatization; 4-((Aminooxy)methyl)-6-chloro-7-hydroxycoumarin; Oxime formation; Monosaccharide
FT-MIR and NIR spectral data fusion: a synergetic strategy for the geographical traceability of Panax notoginseng by Yun Li; Jin-Yu Zhang; Yuan-Zhong Wang (91-103).
Three data fusion strategies (low-llevel, mid-llevel, and high-llevel) combined with a multivariate classification algorithm (random forest, RF) were applied to authenticate the geographical origins of Panax notoginseng collected from five regions of Yunnan province in China. In low-level fusion, the original data from two spectra (Fourier transform mid-IR spectrum and near-IR spectrum) were directly concatenated into a new matrix, which then was applied for the classification. Mid-level fusion was the strategy that inputted variables extracted from the spectral data into an RF classification model. The extracted variables were processed by iterate variable selection of the RF model and principal component analysis. The use of high-level fusion combined the decision making of each spectroscopic technique and resulted in an ensemble decision. The results showed that the mid-level and high-level data fusion take advantage of the information synergy from two spectroscopic techniques and had better classification performance than that of independent decision making. High-level data fusion is the most effective strategy since the classification results are better than those of the other fusion strategies: accuracy rates ranged between 93% and 96% for the low-level data fusion, between 95% and 98% for the mid-level data fusion, and between 98% and 100% for the high-level data fusion. In conclusion, the high-level data fusion strategy for Fourier transform mid-IR and near-IR spectra can be used as a reliable tool for correct geographical identification of P. notoginseng. Graphical abstract The analytical steps of Fourier transform mid-IR and near-IR spectral data fusion for the geographical traceability of Panax notoginseng
Keywords: Panax notoginseng ; Geographical traceability; Data fusion; Fourier transform mid-infrared spectroscopy; Near-infrared spectroscopy
Development of an integrated method of concentration and immunodetection of bacteria by Josune J. Ezenarro; Naroa Uria; Óscar Castillo-Fernández; Noemí Párraga; Miquel Sabrià; Francesc Xavier Muñoz Pascual (105-113).
The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s−1, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase (HRP) bonded to the antibody, and the substrate 3,3′,5,5′-tetramethylbenzidine (TMB). The results showed that the homemade concentration system together with the developed membrane treatment protocol is able to detect E. coli cells with a limit of detection (LoD) of about 100 CFU in 100 mL. Graphical abstract Scheme of the integrated method of concentration and immunodetection of bacteria
Keywords: Microfiltration membrane; Sample concentration; Immunoassay; E. coli ; Pathogen detection
Pattern recognition of 8-hydroxy-2′-deoxyguanosine in biological fluids by Raluca-Ioana Stefan-van Staden; Liliana-Roxana Balahura; Livia Alexandra Gugoasa; Jacobus F. van Staden; Hassan Y. Aboul-Enein; Marcela-Corina Rosu; Stela Maria Pruneanu (115-121).
8-Hydroxy-2′-deoxyguanosine (8-OHdG), a product of oxidative DNA damage, which has been used as a sensitive and reliable marker of oxidative stress and carcinogenesis, is found in high levels in biological fluids of leukemia patients. A reliable screening method based on pattern recognition of 8-OHdG using stochastic sensors designed with graphene materials decorated with nanoparticles of TiO2Ag or TiO2Au was developed. 5,10,15,20-Tetraphenyl-21H,23H porphyrin (P), 2,6-bis((E)-2-(furan-2-yl)vinyl)-4-(4,6,8-trimethylazulen-1-yl)pyridine (Py1), and 2,6-bis((E)-2-(thiophen-3-yl)vinyl)-4-(4,6,8-trimethylazulen-1-yl)pyridine (Py2) were used as modifiers of the graphene paste in the design of sensors. The screening method used for pattern recognition was developed for two pH values accordingly with the nature of the biological fluid to be screened: pH = 3.02 for urine samples and pH = 7.49 for whole blood samples. High sensitivities and low limits of determination for 8-OHdG obtained at both pH values favored the early detection of leukemia. Recoveries over 98.00% with RSD (%) values lower than 1.00% proved the reliability of the proposed screening method. Graphical abstract Graphene based sensors detect 8-hydroxy-2′-deoxyguanosine in biological fluids
Keywords: Pattern recognition; Stochastic microsensors; 8-Hydroxy-2′-deoxyguanosine; Leukemia
Rapid detection of unconjugated estriol in the serum via superparamagnetic lateral flow immunochromatographic assay by Ce Wang; Di Guan; Chen Chen; Shang He; Xiaoting Liu; Chengbin Wang; Huijuan Wu (123-130).
Unconjugated estriol (uE3) is one of the main naturally occurring estrogens that plays an important role in growth and development of the fetus. Usually, the level of uE3 is very low in men and non-pregnant women, but in pregnant women, the level of estriol has been found to be quite high. Therefore, the combination of uE3, AFP, and hCG is now widely used for Down Syndrome screening as a triple marker. Here, we developed a superparamagnetic lateral flow immunochromatographic assay to quantitatively detect uE3. The detection limit of this assay was 0.86 nmol/L and the linear range for the determination of uE3 was from 1 to 100 nmol/L. The detection time was 15 min and the assay had very low cross-reactivity with estrone (E1), estradiol (E2), and progesterone. The coefficient of variation (CV) of intra- and inter-assay ranged from 5% to 13%. The magnetic signals were stable under 37 °C within 7 d. Moreover, the concentrations of uE3 measured by lateral flow immunochromatographic assay in 230 serum samples collected from pregnant women at the Chinese People’s Liberation Army General Hospital had a good correlation with those measured by time-resolved fluorescence immunoassay (R = 0.946).
Keywords: Unconjugated estriol; Lateral flow immunochromatographic assay; Quantitative detection; Superparamagnetic particles; Post of care test (POCT)
Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry by Imeobong U. Antia; Kurian Mathew; Darshna R. Yagnik; Frank A. Hills; Ajit J. Shah (131-143).
Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6–4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA–GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides
Keywords: Procainamide derivatisation; Glycosaminoglycan disaccharides; HILIC UPLC-MS; Heparan sulphate; Cancer cell lines; Selected ion recording (SIR) chromatogram
Short-chained oligo(ethylene oxide)-functionalized gold nanoparticles: realization of significant protein resistance by Kathryn R. Riley; Christopher M. Sims; Imani T. Wood; David J. Vanderah; Marlon L. Walker (145-154).
Protein corona formed on nanomaterial surfaces play an important role in the bioavailability and cellular uptake of nanomaterials. Modification of surfaces with oligoethylene glycols (OEG) are a common way to improve the resistivity of nanomaterials to protein adsorption. Short-chain ethylene oxide (EO) oligomers have been shown to improve the protein resistance of planar Au surfaces. We describe the application of these EO oligomers for improved protein resistance of 30 nm spherical gold nanoparticles (AuNPs). Functionalized AuNPs were characterized using UV-Vis spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. Capillary electrophoresis (CE) was used for separation and quantitation of AuNPs and AuNP-protein mixtures. Specifically, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was employed for the determination of equilibrium and rate constants for binding between citrate-stabilized AuNPs and two model proteins, lysozyme and fibrinogen. Semi-quantitative CE analysis was carried out for mixtures of EO-functionalized AuNPs and proteins, and results demonstrated a 2.5-fold to 10-fold increase in protein binding resistance to lysozyme depending on the AuNP surface functionalization and a 15-fold increase in protein binding resistance to fibrinogen for both EO oligomers examined in this study. Graphical abstract Using capillary electrophoresis, the addition of short-chained oligo(ethylene oxide) ligands to gold nanoparticles was shown to improve protein binding resistance up to 15-fold.
Keywords: Protein corona; Gold nanoparticles; Ethylene oxide; Binding constant; Capillary electrophoresis; NECEEM
New sample preparation method based on task-specific ionic liquids for extraction and determination of copper in urine and wastewater by Tatjana Trtić-Petrović; Aleksandra Dimitrijević; Nikola Zdolšek; Jelena Đorđević; Aleksandar Tot; Milan Vraneš; Slobodan Gadžurić (155-166).
In this study, four hydrophilic ionic liquids (ILs) containing 1-alkyl-3-methylimidazolim cation and either salicylate or chloride anions were synthetized and studied as new task-specific ionic liquids (TSILs) suitable for aqueous biphasic system (ABS) formation and selective one-step extraction of copper(II). TSILs are designed so that the anion is responsible for forming the complex with metal(II) and preventing hydrolysis of metal cations at very strong alkaline pH, whereas the cation is responsible for selective extraction of metal(II)-salicylate complexes. It was found that 1-butyl-3-methylimidazolium salicylate could be used for selective extraction of Cu(II) in the presence of Zn(II), Cd(II), and Pb(II) at very alkaline solution without metal hydroxide formation. It was assumed that formation of metal(II)–salicylate complexes prevents the hydrolysis of the metal ions in alkaline solutions. The determined stability constants for Cu(II)–salicylate complexes, where salicylate was derived from different ionic liquids, indicated that there was no significant influence of the cation of the ionic liquid on the stability of the complexes. The ABS based on 1-butyl-3-methylimidazolium salicylate has been applied as the sample preparation step prior to voltammetric determination of Cu(II). The effect of volume of aqueous sample and IL and extraction time were investigated and optimum extraction conditions were determined. The obtained detection limits were 8 ng dm−3. The optimized method was applied for the determination of Cu(II) in tap water, wastewater, and urine. The study indicated that application of the ABS based on 1-butyl-3-methylimidazolium salicylate ionic liquid could be successfully applied as the sample preparation method for the determination of Cu(II) from various environmental samples. Graphical abstract Aqueous biphasic system based on task-specific ionic liquid as a sample pretreatment for selective determination of Cu(II) in biological and environmental sample
Keywords: Aqueous biphasic systems; Copper(II); Extraction; Task-specific ionic liquids; Sample preparation
Pre-concentration and separation of bacteria by volume coupling electrophoresis on supercritical water-etched fused silica capillary with two segments of different internal diameters and inner surface roughnesses by Marie Horká; Pavel Karásek; Michal Roth; Filip Růžička (167-175).
The transient isotachophoretic stacking and sweeping was used for the on-line large-volume sample pre-concentration of bacteria, Escherichia coli and Staphylococcus aureus cells (methicillin-susceptible or methicillin-resistant), in the initial stage of micellar electrokinetic chromatography using a non-ionogenic surfactant or of capillary electrophoresis, respectively. These procedures were employed in single-piece fused silica capillary etched with supercritical water with two different internal diameter segments featuring different inner surface roughness. Large volumes (maximum 2.8 μL) of the high conductivity sample matrices, physiological saline solution, urine or blood (with purification step), spiked with examined cells were injected into the wider end of a capillary with an inlet inner diameter 195 μm. This novel on-line combination of preconcentration strategies for cells produced an up to 680-fold increase in sensitivity for E. coli or S. aureus cells. The average calculated resolutions, R, for five selected methicillin-susceptible or methicillin-resistant strains were found to be 6.3 for the agar-cultivated and 14.9 for the blood-incubated cells. A low number of bacteria similar to those in clinical samples were also tested. The modified surface roughness step helped to significantly narrow the cell zones and to increase resolution. The migration velocities of E. coli agar-cultivated and blood-incubated cells were approximately the same as those of S. aureus, probably due to the minimal differences in their surface properties. This procedure, on-line pre-concentration and separation of bacteria, is rapid and provides good reproducibility and repeatability.
Keywords: Fused silica capillary; Volume coupling electrophoresis; Supercritical water; Escherichia coli ; Staphylococcus aureus ; Blood
A sediment extraction and cleanup method for wide-scope multitarget screening by liquid chromatography–high-resolution mass spectrometry by Riccardo Massei; Harry Byers; Liza-Marie Beckers; Jens Prothmann; Werner Brack; Tobias Schulze; Martin Krauss (177-188).
Previous studies on organic sediment contaminants focused mainly on a limited number of highly hydrophobic micropollutants accessible to gas chromatography using nonpolar, aprotic extraction solvents. The development of liquid chromatography–high-resolution mass spectrometry (LC–HRMS) permits the spectrum of analysis to be expanded to a wider range of more polar and ionic compounds present in sediments and allows target, suspect, and nontarget screening to be conducted with high sensitivity and selectivity. In this study, we propose a comprehensive multitarget extraction and sample preparation method for characterization of sediment pollution covering a broad range of physicochemical properties that is suitable for LC–HRMS screening analysis. We optimized pressurized liquid extraction, cleanup, and sample dilution for a target list of 310 compounds. Finally, the method was tested on sediment samples from a small river and its tributaries. The results show that the combination of 100 °C for ethyl acetate–acetone (50:50, neutral extract) followed by 80 °C for acetone–formic acid (100:1, acidic extract) and methanol–10 mM sodium tetraborate in water (90:10, basic extract) offered the best extraction recoveries for 287 of 310 compounds. At a spiking level of 1 μg mL-1, we obtained satisfactory cleanup recoveries for the neutral extract—(93 ± 23)%—and for the combined acidic/basic extracts—(42 ± 16)%—after solvent exchange. Among the 69 compounds detected in environmental samples, we successfully quantified several pharmaceuticals and polar pesticides.
Keywords: Sediments; Liquid chromatography; High-resolution mass spectroscopy; Multitarget screening; Pressurized liquid extraction
Comparison of analytical tools appropriate for identification of proteinaceous additives in historical mortars by Iva Krizova; Julia Schultz; Ivan Nemec; Radomir Cabala; Radovan Hynek; Stepanka Kuckova (189-200).
Natural organic additives such as eggs, lard, resins, and oils have been added to mortars since ancient times, because the ancient builders knew of their positive effect on the mortar quality. The tradition of adding organic materials to mortars was commonly handed down only verbally for thousands years. However, this practice disappeared in the nineteenth century, when the usage of modern materials started. Today, one of the most recent topics in the industry of building materials is the reusing of natural organic materials and searching for the forgotten ancient recipes. The research of the old technological approaches involves currently the most advanced analytical techniques and methods. This paper is focussed on testing the possibility of identification of proteinaceous additives in historical mortars and model mortar samples containing blood, bone glue, curd, eggs and gelatine, by Fourier transform infrared (FTIR) and Raman spectroscopy, gas chromatography - mass spectrometry (GC-MS), matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS), liquid chromatography-electrospray ionisation-quadrupole-time of flight mass spectrometry (LC-ESI-Q-TOF MS) and enzyme-linked immunosorbent assay (ELISA). All these methods were applied to the mortar sample taken from the interior of the medieval (sixteenth century) castle in Namest nad Oslavou in the Czech Republic and their comparison contributed to the rough estimation of the protein additive content in the mortar. The obtained results demonstrate that only LC-ESI-Q-TOF MS, MALDI-TOF MS and ELISA have the sufficiently low detection limits that enable the reliable identification of collagens in historical mortars. Graphical abstract Proteomics analyses of historical mortars
Keywords: Protein additives; Historical mortars; FTIR and Raman spectroscopy; Gas chromatography; Mass spectrometry; Immunochemical analysis
A biomimetic approach to conjugate vitamin B6 cofactor with the lysozyme cocooned fluorescent AuNCs and its application in turn-on sensing of zinc(II) in environmental and biological samples by Shilpa Bothra; Lavanya Thilak Babu; Priyankar Paira; SK Ashok Kumar; Rajender Kumar; Suban K Sahoo (201-210).
This communication focusses on the synthesis of red fluorescent lysozyme cocooned gold nanoclusters (Lyso-AuNCs) that have been successfully applied for the selective and specific recognition of the vitamin B6 cofactor pyridoxal-5′-phosphate (PLP). The red fluorescence of Lyso-AuNCs showed remarkable color change to yellow upon conjugation with PLP due to the formation of a Schiff base between the free –NH2 present in the lysozyme and the –CHO group of PLP. The developed PLP conjugated Lyso-AuNCs (PLP_Lyso-AuNCs) was applied for the selective turn-on recognition of Zn2+ ions in aqueous medium. The yellow fluorescence of PLP_Lyso-AuNCs exhibited significant enhancement at 475 nm in the presence of Zn2+ producing bluish-green fluorescence attributed to the complexation-induced aggregation of nanoclusters. The nanoprobe exhibits nanomolar limit of detection for Zn2+ ions (39.2 nM) and the practicality of the nanoprobe was validated in various environmental water samples and biological plasma, urine, and beetroot extract, with fairly good recovery percent. Also, the system was successfully implemented for the intracellular detection and monitoring of Zn2+ in live HeLa cells. Graphical abstract Applications of red emitting lysozyme cocooned gold nanoclusters (Lyso-AuNCs) for the selective recognition of the vitamin B6 cofactor pyridoxal-5′-phosphate (PLP) and the conjugated nano-assembly PLP_Lyso-AuNCs for turn-on detection of Zn2+ ions in various environmental and biological samples.
Keywords: Chemical sensors; Fluorescent gold nanoclusters; Fluorescent turn-on sensor; Live cells imaging; Vitamin B6 cofactor; Zinc(II)
Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR by David Dobnik; Tina Demšar; Ingrid Huber; Lars Gerdes; Sylvia Broeders; Nancy Roosens; Frederic Debode; Gilbert Berben; Jana Žel (211-221).
Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison
Keywords: Digital PCR; Droplet digital PCR; Absolute quantification; Reference materials; GMO quantification
A multiplex immunochromatographic test using gold nanoparticles for the rapid and simultaneous detection of four nitrofuran metabolites in fish samples by Quan Wang; Yingchun Liu; Mingyan Wang; Yongjun Chen; Wei Jiang (223-233).
There is an urgent need for the rapid and simultaneous detection of multiple analytes present in a sample matrix. Here, a multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites, i.e., 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), in fish samples. Four different antigens were separately immobilized in four test lines on a nitrocellulose membrane. Goat anti-mouse immunoglobulin (IgG) was used as a control. Sensitive and specific monoclonal antibodies (mAbs) that recognize the corresponding antigens were selected for the assay, and no cross-reactivity between the antibodies in the detection assay was observed. The free analytes in samples or standards were pre-incubated with freeze-dried mAb–gold conjugates to improve the sensitivity of the detection assay. The multi-ICT detection was accomplished in less than 15 min by the naked eye. The cutoff values for the strip test were 0.5 ng/mL for AOZ and 0.75 ng/mL for AHD, SEM, and AMOZ, which were all below the maximum residue levels set by the European Union and China. A high degree of consistency was observed between the multi-ICT method and commercially available enzyme-linked immunosorbent assay (ELISA) kits using spiked, incurred, and “blind” fish samples, indicating the accuracy, reproducibility, and reliability of the novel test strip. This newly developed multi-ICT strip assay is suitable for the rapid and high-throughput screening of four nitrofuran metabolites in fish samples on-site, with no treatment or devices required. Graphical abstract A multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites (AOZ, SEM, AMOZ, and AHD) in fish samples
Keywords: Multiplexed immunoassay; Simultaneous detection; Immunochromatographic test strip; Nitrofuran metabolites
Optimization of ultrahigh-speed multiplex PCR for forensic analysis by Georgiana Gibson-Daw; Karin Crenshaw; Bruce McCord (235-245).
In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl2, and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis
Keywords: Forensic; Genotyping; Rapid PCR; Short tandem repeat
Synthesis of multirecognition magnetic molecularly imprinted polymer by atom transfer radical polymerization and its application in magnetic solid-phase extraction by Xiang-Jin Kong; Chao Zheng; Yao-Han Lan; Shuai-Shuai Chi; Qian Dong; Hao-Long Liu; Chao Peng; Lin-Yi Dong; Liang Xu; Xian-Hua Wang (247-257).
In this work, we reported an effective method for the synthesis of a multirecognition magnetic molecularly imprinted polymer (MMIP) with atom transfer radical polymerization (ATRP), using 2,4-diamino-6-methyl-1,3,5-triazine as pseudo-template. The resulting MMIP was characterized in detail by Fourier transform-infrared (FT-IR) spectra, scanning electron microscopy (SEM), thermogravimetic analysis (TGA), and vibrating sample magnetometry (VSM). These results indicated the successful synthesis of MMIP with sufficient thermal stability and magnetic properties. The adsorption experiments were carried out to evaluate the specific selectivity of MMIP related to the spatial structure of target molecules. The MMIP exhibited multirecognition ability and excellent binding capability for melamine (MEL), cyromazine (CYR), triamterene (TAT), diaveridine (DVD), and trimethoprim (TME), and the apparent maximum number of binding sites (Q max) was 77.5, 75.2, 72.5, 69.9, and 70.4 μmol g−1, respectively. The multirecognition MMIP not only possessed adequate magnetic responsiveness for fast separation but also avoided the risk of template leakage on trace component analysis. Therefore, it was suitable for serving as a magnetic solid-phase extraction (MSPE) adsorbent. MSPE coupled with high-performance liquid chromatography analysis was applied to enrich and separate five target molecules from three samples. Recoveries for all target molecules ranged from 81.6 to 91.5% with relative standard deviations of no more than 4.1% (n = 3). Graphical abstract Multirecognition property of magnetic molecularly imprinted polymer prepared with pseudo template
Keywords: Multirecognition; Magnetic molecularly imprinted polymer; Atom transfer radical polymerization; Magnetic solid-phase extraction
Steroid hormone profiling in human breast adipose tissue using semi-automated purification and highly sensitive determination of estrogens by GC-APCI-MS/MS by Kristin Hennig; Jean Philippe Antignac; Emmanuelle Bichon; Marie-Line Morvan; Isabelle Miran; Suzette Delaloge; Jean Feunteun; Bruno Le Bizec (259-275).
Body mass index is a known breast cancer risk factor due to, among other mechanisms, adipose-derived hormones. We developed a method for steroid hormone profiling in adipose tissue to evaluate healthy tissue around the tumor and define new biomarkers for cancer development. A semi-automated sample preparation method based on gel permeation chromatography and subsequent derivatization with trimethylsilyl (TMS) is presented. Progestagens and androgens were determined by GC-EI-MS/MS (LOQ 0.5 to 10 ng/g lipids). For estrogen measurement, a highly sensitive GC-APCI-MS/MS method was developed to reach the required lower limits of detection (0.05 to 0.1 ng/g lipids in matrix, 100–200 fg on column for pure standards). The combination of the two methods allows the screening of 27 androgens and progestagens and 4 estrogens from a single sample. Good accuracies and repeatabilities were achieved for each compound class at their respective limit of detection. The method was applied to determine steroid hormone profiles in adipose tissue of 51 patients, collected both at proximity and distant to the tumor. Out of the 31 tested steroid hormones, 14 compounds were detected in human samples. Pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone (DHEA), and androstendione accounted together for 80% of the observed steroid hormone profiles, whereas the estrogens accounted for only 1%. These profiles did not differ based on sampling location, except for ß-estradiol; steroid hormone conversions from androgens to estrogens that potentially take place in adipose or tumoral tissue might not be detectable due a factor 100 difference in concentration of for example DHEA and ß-estradiol. Graphical Abstract Schematic overview of the determination of steroid hormones and metabolites in adipose tissue in proximity and distal to the tumor
Keywords: Steroid hormones; Breast cancer; Adipose tissue; GC-APCI-MS/MS; Atmospheric pressure chemical ionization; Mass spectrometry; Gas chromatography
Combining Raman and laser induced breakdown spectroscopy by double pulse lasing by Vasily N. Lednev; Sergey M. Pershin; Pavel A. Sdvizhenskii; Mikhail Ya. Grishin; Alexander N. Fedorov; Vladimir V. Bukin; Vadim B. Oshurko; Alexander N. Shchegolikhin (277-286).
A new approach combining Raman spectrometry and laser induced breakdown spectrometry (LIBS) within a single laser event was suggested. A pulsed solid state Nd:YAG laser running in double pulse mode (two frequency-doubled sequential nanosecond laser pulses with dozens microseconds delay) was used to combine two spectrometry methods within a single instrument (Raman/LIBS spectrometer). First, a low-energy laser pulse (power density far below ablation threshold) was used for Raman measurements while a second powerful laser pulse created the plasma suitable for LIBS analysis. A short time delay between two successive pulses allows measuring LIBS and Raman spectra at different moments but within a single laser flash-lamp pumping. Principal advantages of the developed instrument include high quality Raman/LIBS spectra acquisition (due to optimal gating for Raman/LIBS independently) and absence of target thermal alteration during Raman measurements. A series of high quality Raman and LIBS spectra were acquired for inorganic salts (gypsum, anhydrite) as well as for pharmaceutical samples (acetylsalicylic acid). To the best of our knowledge, the quantitative analysis feasibility by combined Raman/LIBS instrument was demonstrated for the first time by calibration curves construction for acetylsalicylic acid (Raman) and copper (LIBS) in gypsum matrix. Combining ablation pulses and Raman measurements (LIBS/Raman measurements) within a single instrument makes it an efficient tool for identification of samples hidden by non-transparent covering or performing depth profiling analysis including remote sensing. Graphical abstract Combining Raman and laser induced breakdown spectroscopy by double pulse lasing
Keywords: Laser induced breakdown spectroscopy; LIBS; Raman scattering; Double pulse; Quantitative analysis
Universal fluorometric aptasensor platform based on water-soluble conjugated polymers/graphene oxide by Limin Guo; You Hu; Ziqi Zhang; Yanli Tang (287-295).
We designed a universal and sensitive fluorometric aptasensor that uses a fluorescence resonance energy transfer (FRET) mechanism. In the aptasensor, water-soluble conjugated poly(9,9-bis(6′-N,N,N-trimethylammonium)hexyl)fluorine phenylene (PFP) is used as the energy donor and a carboxyfluorescein (FAM)-labeled aptamer is used as the energy acceptor. Graphene oxide (GO) used as a quencher can specifically adsorb the aptamer, leading to quenching of the FAM fluorescence. In the presence of targets, the aptamer can change its conformation to prevent adsorption by GO. Strong FRET was thus obtained owing to the electrostatic interactions between PFP and the aptamer. In contrast, in the absence of targets, the FRET was weak because of GO specifically adsorbing the aptamer and quenching the fluorescence. Bisphenol A (a pollutant molecule) and dopamine (a biomolecule) were used as models to successfully validate the feasibility, universality, and high selectivity and sensitivity of this aptasensor. This method can detect BPA at environmentally relevant concentrations (less than 1 ng/mL) with a limit of detection of 0.005 ng/mL. A low limit of detection (1.0 nmol/L) was also obtained for dopamine. In addition, this aptasensor is applicable in real samples and in diluted human plasma and human serum. Good recovery rates from 95% to 105% and from 95% to 107% were obtained for bisphenol A and dopamine, respectively. Furthermore, adenosine detection was successfully achieved by the same mechanism, proving the universality. It is expected that the aptasensor could be applied in detecting other contaminants, biomolecules, and heavy metal ions by a change in only the aptamer sequence. Graphical abstract A universal and sensitive fluorometric aptasensor was developed for the detection of small molecules that uses a fluorescence resonance energy transfer (FRET) mechanism. GO graphene oxide. PFP poly(9,9-bis(6′-N,N,N-trimethylammonium)hexyl)fluorine phenylene
Keywords: Water-soluble conjugated polymers; Aptasensor; Graphene oxide; Fluorescence resonance energy transfer; Bisphenol A; Dopamine