Analytical and Bioanalytical Chemistry (v.409, #28)

Exhaled breath condensate methods adapted from human studies using longitudinal metabolomics for predicting early health alterations in dolphins by Eva Borras; Alexander A. Aksenov; Mark Baird; Brittany Novick; Michael Schivo; Konstantin O. Zamuruyev; Alberto Pasamontes; Celeste Parry; Soraya Foutouhi; Stephanie Venn-Watson; Bart C. Weimer; Cristina E. Davis (6523-6536).
is a postdoctoral fellow in the BioMEMS and Bioinstrumentation Group in the Department of Mechanical and Aerospace Engineering at the University of California, Davis. Her current research is focused on breath metabolomic studies using mass spectrometry and data analysis. earned his PhD degree in chemistry from the University of Florida in 2006, and received postdoctoral training in mass spectrometry at Carnegie Mellon University. He moved to the University of California, Davis in 2010 as an associate specialist. His expertise is in bioanalytical chemistry and mass spectrometry, and his primary research activities and interests are in metabolomics, biological research, and various aspects of basic and applied bioanalytical chemistry. He is currently a project scientist at the University of California, San Diego. is a veterinary technician with the National Marine Mammal Foundation. He assists with various research projects and helps provide care for the animals of the US Navy Marine Mammal Program. earned her BS degree in marine and aquatic biology from Bowling Green State University. She is currently a veterinary research technician at the National Marine Mammal Foundation in San Diego, California, and plans to attend veterinary school. is a clinician-scientist who conducts clinical studies that utilize the collection and study of breath metabolites. He works at the interface of clinical medicine and engineering to advance the design and use of novel diagnostic platforms. received his PhD degree from the Department of Mechanical and Aerospace Engineering at the University of California, Davis. His research specializes in fabrication and development of microfluidic systems and bioinstruments for liquid and gas analyses. was an associate specialist in the BioMEMS and Bioinstrumentation Group in the Department of Mechanical and Aerospace Engineering at the University of California, Davis. His research was focused on metabolomics doing preprocessing and data analysis. He is presently a researcher at the Leiden Academic Center for Drug Research, Division of Analytical BioSciences. is the clinical research manager for the National Marine Mammal Foundation. Her recent research has focused on marine mammal metabolism and infectious diseases. She also manages the biological sample archive and medical record database as part of her work with the US Navy Marine Mammal Program. completed her MS and PhD degrees in comparative pathology at the University of California, Davis. Her work focused on host-pathogen interactions and mesenchymal stem cell metabolism. This was followed by a postdoctoral fellowship in the fields of genomics and metabolomics. She is currently pursuing an MD degree at Tulane University. is Director of Translational Medicine and Research at the National Marine Mammal Foundation. Her research has focused on aging-associated metabolic conditions present in dolphins and humans, including metabolic syndrome and dysmetabolic iron overload syndrome. is a professor in the Department of Population Health and Reproduction in the UC Davis School of Veterinary Medicine at the University of California, Davis. His laboratory focuses on microbial physiology and function using systems biology approaches in food, animals, and the environment to examine the interaction of pathogens and host association. is Vice Chair and Professor of Mechanical and Aerospace Engineering at the University of California, Davis. She is a Fellow of the American Institute for Medical and Biological Engineering and a member of the Scientific Advisory Board for the US Air Force. Her research focuses on metabolomics and novel chemical sensor designs. Monitoring health conditions is essential to detect early asymptomatic stages of a disease. To achieve this, blood, urine and breath samples are commonly used as a routine clinical diagnostic. These samples offer the opportunity to detect specific metabolites related to diseases and provide a better understanding of their development. Although blood samples are commonly used routinely to monitor health, the implementation of a relatively noninvasive technique, such as exhaled breath condensate (EBC) analysis, may further benefit the well-being of both humans and other animals. EBC analysis can be used to track possible physical or biochemical alterations caused by common diseases of the bottlenose dolphin (Tursiops truncatus), such as infections or inflammatory-mediated processes. We have used an untargeted metabolomic method with liquid chromatography–mass spectrometry analysis of EBC samples to determine biomarkers related to disease development. In this study, five dolphins under human care were followed up for 1 year. We collected paired blood, physical examination information, and EBC samples. We then statistically correlated this information to predict specific health alterations. Three dolphins provided promising case study information about biomarkers related to cutaneous infections, respiratory infections, dental disease, or hormonal changes (pregnancy). The use of complementary liquid chromatography platforms, with hydrophilic interaction chromatography and reverse-phased columns, allowed us to detect a wide spectrum of EBC biomarker compounds that could be related to these health alterations. Moreover, these two analytical techniques not only provided complementary metabolite information but in both cases they also provided promising diagnostic information for these health conditions. Graphical abstract Collection of the exhaled condensed breath from a bottlenose dolphin from U.S. Navy Marine Mammal Program (MMP)
Keywords: Exhaled breath condensate; Breath analysis; Metabolomics; Longitudinal study; Liquid chromatography–mass spectrometry

Accurate and reliable quantification of endogenous lipid mediators in complex biological samples is a daunting challenge. In this study, a robust and direct endogenous quantitative method using background subtracting calibration curves by liquid chromatography-tandem mass spectrometry was first developed for the determination of endogenous lipid mediators in ischemic stroke rats. Absolute quantification without surrogate matrix could be achieved by using background subtracting calibration curves, which were corrected and verified from standard curves constructed on original matrix. The recoveries of this method were in the range of 50.3–98.3%, the precision with the relative standard deviation was less than 13.8%, and the accuracy with the relative error was within ± 15.0%. In addition, background subtracting calibration curves were further verified by validation factors ranging from 90.3 to 110.9%. This validated method has been successfully applied to the analysis of seven endogenous inflammation-related lipid mediators in the brain tissues of ischemic stroke rats. The results indicated that prostaglandins as inflammatory factors and some lipid mediators with neuroprotective effects increased apparently (p < 0.05) in the stroke groups compared with the normal rats. Besides, the two drugs (isosteviol sodium and edaravone) could significantly reduce (p < 0.05) the levels of prostaglandin E2 and prostaglandin F of stroke rats to inhibit inflammation. Based on the results, it is strongly believed that this approach can be readily generalized as a new reference for the quantification of endogenous compounds in the complex biological samples. Graphical abstract The analysis procedure of determining endogenous inflammation-related lipid mediators using BSCC by LC-MS/MS
Keywords: Endogenous quantification; Background subtracting calibration curve (BSCC); Lipid mediators; Mass spectrometry

Apparent activation energies of protein–protein complex dissociation in the gas–phase determined by electrospray mass spectrometry by Yelena Yefremova; F. Teresa I. Melder; Bright D. Danquah; Kwabena F.M. Opuni; Cornelia Koy; Alexandra Ehrens; David Frommholz; Harald Illges; Knut Koelbel; Frank Sobott; Michael O. Glocker (6549-6558).
We have developed a method to determine apparent activation energies of dissociation for ionized protein–protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein–protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation ( E A m 0 g # $$ {E}_{A m0g}^{#} $$ ) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein–protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants. Graphical abstract Electrospray mass spectrometry and collision-induced dissociation delivers apparent activation energies and supramolecular bond force constants of protein-protein complexes in the gas phase.
Keywords: Protein–protein interaction; Native mass spectrometry; Ion mobility; Collision induced dissociation; Quasi equilibrium conditions

Porcine relaxin is a 6 kDa peptide hormone of pregnancy with important physiological and pharmacological effects. It contains a number of analogs of which porcine relaxin B29 is one of the most important. To support the development of porcine relaxin B29 as a new drug, we established an UPLC-MS/MS method for its quantitation in dog plasma. Sample preparation by protein precipitation and ion exchange solid phase extraction was followed by UPLC on an XBridge™ BEH300 C18 column at 40 °C in a run time of only 5.5 min. Detection was performed on a Qtrap 6500 mass spectrometer using ESI in the positive ion mode with MRM of the transitions at m/z 831.7 [M+7H]7+ → 505.4 and m/z 1162.4 [M+5H]5+ → 226 for pRLX B29 and internal standard (recombinant human insulin), respectively. The method was linear over the concentration range 30–2000 ng/mL with no matrix effects. Intra- and inter-day precisions were < 15% with accuracies in the range 98.8–100.6%. The method was successfully applied to a pharmacokinetic study in beagle dogs after administration of a 0.15 mg/kg intravenous dose. Graphical abstract Sample preparation and detection procedure.
Keywords: Ion exchange solid phase extraction; LC-MS/MS; Porcine relaxin; Ultrahigh performance liquid chromatography

A smartphone colorimetric reader integrated with an ambient light sensor and a 3D printed attachment for on-site detection of zearalenone by Yuan Chen; Qiangqiang Fu; Dagang Li; Jun Xie; Dongxu Ke; Qifang Song; Yong Tang; Hong Wang (6567-6574).
Smartphone biosensors could be cost-effective, portable instruments to be used for the readout of liquid colorimetric assays. However, current reported smartphone colorimetric readers have relied on photos of liquid assays captured using a camera, and then analyzed using software programs. This approach results in a relatively low accuracy and low generality. In this work, we reported a novel smartphone colorimetric reader that has been integrated with an ambient light sensor and a 3D printed attachment for the readout of liquid colorimetric assays. The portable and low-cost ($0.15) reader utilized a simplified electronic and light path design. Furthermore, our reported smartphone colorimetric reader can be compatible with different smartphones. As a proof of principle, the utility of this device was demonstrated using it in conjunction with an enzyme-linked immunosorbent assay to detect zearalenone. Results were consistent with those obtained using a professional microplate reader. The developed smartphone colorimetric reader was capable of providing scalable, cost-effective, and accurate results for liquid colorimetric assays that related to clinical diagnoses, environment pollution, and food testing. Graphical abstract A novel smartphone colorimetric reader that has been integrated with an ambient light sensor and a 3D printed attachment for the readout of liquid colorimetric assays.
Keywords: Smartphone; Zearalenone; Enzyme-linked immunosorbent assays; Colorimetric reader; Ambient light sensor

Study on the antigenicity of metallofullerenol: antibody production, characterization, and its enzyme immunoassay application by Xihong Guo; Shangyuan Yang; Huan Huang; Rongli Cui; Cheng Li; Huanli Yao; Bing Liu; Lele Zhang; Binggang Xu; Jinquan Dong; Baoyun Sun (6575-6581).
With their intriguing structures and properties, metallofullerenols have attracted considerable attention in biological and medical applications. Due to the increasing biomedical interest, effective detection methods are important to monitor and control metallofullerenols. However, the detection of metallofullerenols becomes very difficult after polyhydroxylated modification due to the lack of detectable features. Antibody-based immunoassay methods have been important tools for detection and will better meet the needs of analysis of metallofullerenols. Thus, the antigenicity of metallofullerenol has been studied for the first time. In this study, no immune response was detected when metallofullerenol Gd@C82(OH)x was used as immunogen. However, the polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen, indicating that metallofullerenol can act as hapten. The specificity of the obtained antibody was investigated. It has been found that the hydroxyl groups on the surface of the carbon cage, the encapsulated metal, and the size and shape of the carbon cage did not affect the recognition specificity of the antibody. Based on the obtained antibody, an indirect competitive enzyme immunoassay was developed for the determination of metallofullerenol with detection limits of 18 ng/mL in PBS. This enzyme immunoassay method was successfully used to detect metallofullerenol in serum. This work can provide an innovative way to determine metallofullerenols. Graphical abstract The polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen. Based on the obtained antibody, a competitive enzyme immunoassay was developed for the determination of metallofullerenol.
Keywords: Metallofullerene; Enzyme immunoassay; Polyclonal antibody; Immunogen; Nanomedicine

Development of an LC-MS/MS method with protein G purification strategy for quantifying bevacizumab in human plasma by Huai-Hsuan Chiu; I-Lin Tsai; Yen-Shen Lu; Ching-Hung Lin; Ching-Hua Kuo (6583-6593).
Biopharmaceutical products such as protein drugs and monoclonal antibodies (mAb) are currently of great interest with monoclonal antibody drugs being one of the fastest growing categories of biopharmaceutical products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has gained high interest for measuring mAb drugs in biological samples in recent years due to its high selectivity. Bevacizumab is a humanized immunoglobulin G (IgG) mAb drug against human vascular endothelial cell growth factor A (VEGF-A). It is used for treating many types of cancers. Recent studies have indicated that clinical outcomes vary among patients treated with bevacizumab and produce various side effects, such as vascular disorders. In this study, we developed an LC-MS/MS method to quantify bevacizumab concentration. We selected readily available and economic materials for sample preparation to facilitate its wider use in clinical fields.—Protein G was used to trap bevacizumab from human plasma. In place of an extended stable isotope-labeled internal standard (SIL-IS), the IgG-based drug-IS tocilizumab was used because of its better calibration performance. The method was validated in terms of its precision, accuracy, linearity, and sensitivity. The accuracies which were expressed as percentage recoveries for three concentration levels were within 92.8 ± 3.2 to 112.7 ± 4.5%. Repeatability and intermediate precision in terms of peak area ratios were lower than 5.2 and 12.9% RSD, respectively. The application to patients’ sample measurements revealed a wide individual variability of drug concentrations, and the proposed simple and general method may facilitate personalized medicine for improving therapeutic efficacy and safety. Graphical abstract ᅟ
Keywords: Bevacizumab; Protein G purification; Monoclonal antibody (mAb); Plasma; In-solution digestion; LC-MS/MS

Identification of terpenes and essential oils by means of static headspace gas chromatography-ion mobility spectrometry by Roman Rodríguez-Maecker; Eduardo Vyhmeister; Stefan Meisen; Antonio Rosales Martinez; Andriy Kuklya; Ursula Telgheder (6595-6603).
Static headspace gas chromatography-ion mobility spectrometry (SHS GC-IMS) is a relatively new analytical technique that has considerable potential for analysis of volatile organic compounds (VOCs). In this study, SHS GC-IMS was used for the identification of the major terpene components of various essential oils (EOs). Based on the data obtained from 25 terpene standards and 50 EOs, a database for fingerprint identification of characteristic terpenes and EOs was generated utilizing SHS GC-IMS for authenticity testing of fragrances in foods, cosmetics, and personal care products. This database contains specific normalized IMS drift times and GC retention indices for 50 terpene components of EOs. Initially, the SHS GC-IMS parameters, e.g., drift gas and carrier gas flow rates, drift tube, and column temperatures, were evaluated to determine suitable operating conditions for terpene separation and identification. Gas chromatography-mass spectrometry (GC-MS) was used as a reference method for the identification of terpenes in EOs. The fingerprint pattern based on the normalized IMS drift times and retention indices of 50 terpenes is presented for 50 EOs. The applicability of the method was proven on examples of ten commercially available food, cosmetic, and personal care product samples. The results confirm the suitability of SHS GC-IMS as a powerful analytical technique for direct identification of terpene components in solid and liquid samples without any pretreatment. Graphical abstract Fingerprint pattern identification of terpenes and essential oils using static headspace gas chromatography-ion mobility spectrometry.
Keywords: Static headspace (SHS); Gas chromatography (GC); Ion mobility spectrometry (IMS); Essential oils (EOs); Terpenes; Fingerprint identification

Terbium chelation, a specific fluorescent tagging of human transferrin. Optimization of conditions in view of its application to the HPLC analysis of carbohydrate-deficient transferrin (CDT) by Silvia Nicotra; Daniela Sorio; Giulia Filippi; Luca De Gioia; Veronica Paterlini; Elio Franco De Palo; Rita Grandori; Franco Tagliaro; Carlo Santambrogio (6605-6612).
Transferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+ complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), “native” mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+ complex, yielding a specific signature. The interaction of Tb3+ with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+ complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+ or Fe3+ binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving relevant increase (by a factor of ∼3) of analytical sensitivity. Graphical abstract Schematic representation of HPLC-separated transferrin glycoforms detected by fluorescence emission of the terbium ions bound to the protein.
Keywords: Alcohol abuse; Carbohydrate-deficient transferrin; Terbium fluorescence; Capillary electrophoresis; High-performance liquid chromatography; Protein-metal interaction

Elastic and inelastic light scattering spectroscopy and its possible use for label-free brain tumor typing by Edwin Ostertag; Mona Stefanakis; Karsten Rebner; Rudolf W. Kessler (6613-6623).
This paper presents an approach for label-free brain tumor tissue typing. For this application, our dual modality microspectroscopy system combines inelastic Raman scattering spectroscopy and Mie elastic light scattering spectroscopy. The system enables marker-free biomedical diagnostics and records both the chemical and morphologic changes of tissues on a cellular and subcellular level. The system setup is described and the suitability for measuring morphologic features is investigated. Graphical Abstract Bimodal approach for label-free brain tumor typing. Elastic and inelastic light scattering spectra are collected laterally resolved in one measurement setup. The spectra are investigated by multivariate data analysis for assigning the tissues to specific WHO grades according to their malignancy
Keywords: Mie elastic light scattering spectroscopy; Raman inelastic light scattering spectroscopy; Bimodal; Multi-spectroscopy; Label-free; Brain tumor typing

Microchip capillary gel electrophoresis combined with lectin affinity enrichment employing magnetic beads for glycoprotein analysis by Nicole Y. Engel; Victor U. Weiss; Christian Wenz; Susanne Glück; Andreas Rüfer; Martin Kratzmeier; Martina Marchetti-Deschmann; Günter Allmaier (6625-6634).
Due to the constant search for reliable methods to investigate glycoproteins in complex biological samples, an alternative approach combining affinity enrichment with rapid and sensitive analysis on-a-chip is presented. Glycoproteins were specifically captured by lectin-coated magnetic beads, eluted by competitive sugars, and investigated with microchip capillary gel electrophoresis (MCGE), i.e., CGE-on-a-chip. We compared our results to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, which turned out to be in very good agreement. While SDS-PAGE offers the possibility of subsequent mass spectrometric analysis of captured and separated analytes, MCGE scores with time savings, higher throughput, and lower sample consumption as well as quality control (QC) and process analytical technology (PAT) applicability. Due to these advantages, a lectin-based glycoprotein capture protocol can easily be optimized. In our case, two different types of magnetic beads were tested and compared regarding lectin binding. The selectivity of our strategy was demonstrated with a set of model glycoproteins, as well as with human serum and serum depleted from high-abundance proteins. The specificity of the capturing method was investigated revealing to a certain degree an unspecific binding between each sample and the beads themselves, which has to be considered for any specific enrichment and data interpretation. In addition, two glycoproteins from Trichoderma atroviride, a fungus with mycoparasitic activity and only barely studied glycoproteome, were enriched by means of a lectin and so identified for the first time. Graphical abstract Glycoproteins from biological samples were detected by microchip capillary gel electrophoresis after lectin affinity enrichment using magnetic beads and elution with respective competitive monosaccharides
Keywords: Affinity enrichment; Glycoprotein; Lab-on-a-chip; Lectin; Magnetic beads

A microfluidics-based mobility shift assay to identify new inhibitors of β-secretase for Alzheimer’s disease by Rongfeng Liu; Yu-Chih Liu; Junwei Meng; Haiyan Zhu; Xuehong Zhang (6635-6642).
The β-secretase (BACE1) initiates the generation of toxic amyloid-β peptide (Aβ) from amyloid-β precursor protein (APP), which was widely considered to play a key role in the pathogenesis of Alzheimer’s disease (AD). Here, a novel microfluidics-based mobility shift assay (MMSA) was developed, validated, and applied for the screening of BACE1 inhibitors for AD. First, the BACE1 activity assay was established with a new fluorescent peptide substrate (FAM-EVNLDAEF) derived from the Swedish mutant APP, and high-quality ratiometric data were generated in both endpoint and kinetic modes by electrophoretic separation of peptide substrate from the BACE1 cleaved product (FAM-EVNL) before fluorescence quantification. To validate the assay, the inhibition and kinetic parameter values of two known inhibitors (AZD3839 and AZD3293) were evaluated, and the results were in good agreement with those reported by other methods. Finally, the assay was applied to screen for new inhibitors from a 900-compound library in a 384-well format, and one novel hit (IC50 = 26.5 ± 1.5 μM) was identified. Compared with the common fluorescence-based assays, the primary advantage of the direct MMSA was to discover novel BACE1 inhibitors with lower auto-fluorescence interference, and its superb capability for kinetic study. Graphical abstract Microfluidics-based mobility shift assay for BACE1.
Keywords: Alzheimer’s disease; Amyloid-β peptide; β-Secretase; BACE1; Mobility shift assay; Kinetic study

A novel adsorbent based on the surface of magnetic graphene oxide-grafted cellulose nanocrystal molecularly imprinted polymers (Mag@GO-g-CNCs@MIPs) was developed for the selective extraction and fast adsorption of fluoroquinolones (FQs) from river water samples. Cellulose nanocrystals (CNCs) were grafted onto activated graphene oxide (GO), and the surfaces of the obtained magnetic GO-g-CNC particles were molecularly imprinted with polymers using ofloxacin (OFX) as a template molecule and methacrylic acid (MAA) as a functional monomer. The resulting Mag@GO-g-CNCs@MIP material was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy, vibrating sample magnetometry, and X-ray photoelectron spectroscopy. Under optimum adsorption conditions, the Mag@GO-g-CNCs@MIPs with large specific surface area were easily collected and separated using an external magnetic field. Mag@GO-g-CNCs@MIPs exhibited an ultra-fast adsorption profile for FQs (5 min to achieve the maximum adsorption capacity of 74 mg/g), with imprinting factor values ranging from 1.5 to 3.1. High recognition selectivity towards nine FQs from real river water samples was established through coupling with high-performance liquid chromatography (HPLC), and the recovery of samples spiked with nine FQs was found to be in the range of 79.2–96.1%, with a detection limit ranging from 6.5 to 51 ng/g. Moreover, the data obtained adhered to the Freundlich isotherm model, and the adsorption kinetics followed a pseudo-second-order model. Finally, the Mag@GO-g-CNCs@MIPs could be regenerated and reused for seven consecutive cycles with only a 13% drop in adsorption capacity, indicating its effective application as a new, reusable, and selective adsorbent for the enrichment and separation of FQs from aqueous solutions.
Keywords: Graphene oxide; Cellulose nanocrystals; Fluoroquinolones; Surface grafting; Molecularly imprinted polymers

Ratiometric fluorescent sensing of copper ion based on chromaticity change strategy by Hongmei Li; Xuefeng Wang; Zheng Cai; Ling Lu; Jia Tao; Bin Sun; Yuanyuan Yang; Quan Xu; Zhiqiang Yu; Peng Zhao (6655-6662).
Copper ions play a significant role in human bodies, as they are beneficial for many metabolism processes, but excessive Cu2+ may cause various symptoms. Therefore, the development of strategies capable of both sensitive detection and visualization of Cu2+ is greatly important for human health. In this work, a novel ratiometric fluorescence sensor for rapid and visual detection of Cu2+ was designed based on green emission carbon dots (CDs) and red emission CdTe/CdS quantum dots (QDs). We have calculated the chromaticity coordinates of individual CDs and CdTe/CdS QDs, and the chromaticity coordinate of their mixture would change along the line between the two points of CDs and CdTe/CdS QDs in the chromaticity coordinate system. The red fluorescence of QDs can be influenced by Cu2+, and the fluorescence color of CDs-QDs changes from yellow to green as the chromaticity coordinates change with the addition of Cu2+. This traffic-light-type color change was more sensitive to the naked eye compared to conventional single emission-based sensors. This paper not only realized ratiometric fluorescence and visual detection of Cu2+ through a convenient method but also provided a promising strategy of designing an assay kit for trace Cu2+. Graphical abstract Ratiometric fluorescent sensing of copper ion based on chromaticity change strategy
Keywords: Chromaticity coordinate; Visual detection; Carbon dots; Quantum dots; Real samples

A reliable protocol for colorimetric determination of iron oxide nanoparticle uptake by cells by Daiana K. Deda; Roberta M. Cardoso; Mayara K. Uchiyama; Christiane Pavani; Sergio H. Toma; Mauricio S. Baptista; Koiti Araki (6663-6675).
Size, shape, and surface properties of superparamagnetic iron oxide nanoparticles (SPIONs) can influence their interaction with biological systems, particularly the incorporation by tumor cells and consequently the biological activity and efficiency in biomedical applications. Several strategies have been used to evaluate cellular uptake of SPIONs. While qualitative methods are generally based on microscopy techniques, quantitative assays are carried out by techniques such as inductively coupled plasma-mass spectrometry and flow cytometry. However, inexpensive colorimetric methods based on equipments commonly found in chemistry and biochemistry laboratories are preferred for routine measurements. Nevertheless, colorimetric assays must be used judiciously, particularly when nanoparticles are involved, since their interaction with biological constituents tends to lead to quite underestimated results. Thus, herein described is a colorimetric protocol using 2,2′-bipyridine as chromogenic ligand, where each step was optimized and validated by total reflection X-ray fluorescence spectroscopy, realizing a highly reproducible and reliable method for determination of iron content in cells incubated with SPIONs. The limit of blank and limit of detection were determined to be as low as 0.076 and 0.143 μg Fe/mL, using sample volumes as small as 190 μL and a number of cells as low as 2.0 × 105. Furthermore, three different types of surface-functionalized nanoparticles were incorporated in cells and evaluated through this protocol, enabling to monitor the additive effect of o-phosphorylethanolamine (PEA) and folic acid (FA) conjugation on iron oxide nanoparticles (SPION-PEA and SPION-PEA/FA), that enhanced the uptake by HeLa cells, respectively, by four and ten times when compared to SPIONs conjugated with nonbioactive molecules. Graphical abstract Colorimetric determination of superparamagnetic iron oxide nanoparticles (SPIONs) incorporated by cells.
Keywords: Iron analyses; Iron oxide nanoparticles; Cellular uptake; Colorimetric analyses; X-ray fluorescence

Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters by Yufang Hu; Qingqing Zhang; Lihua Xu; Jiao Wang; Jiajia Rao; Zhiyong Guo; Sui Wang (6677-6688).
Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient “signal-on” electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3’-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H2O2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3′-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.
Keywords: Electrochemical biosensor ; Terminal deoxynucleotidyl transferase; BamHI; Copper nanoclusters; Graphene oxide

Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR by Alexandra Bogožalec Košir; Carla Divieto; Jernej Pavšič; Stefano Pavarelli; David Dobnik; Tanja Dreo; Roberto Bellotti; Maria Paola Sassi; Jana Žel (6689-6697).
Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
Keywords: Droplet digital PCR; Droplet volume; DNA quantification; Optical microscopy imaging

Estimation of start and stop numbers for cluster resolution feature selection algorithm: an empirical approach using null distribution analysis of Fisher ratios by Lawrence A. Adutwum; A. Paulina de la Mata; Heather D. Bean; Jane E. Hill; James J. Harynuk (6699-6708).
Cluster resolution feature selection (CR-FS) is a hybrid feature selection algorithm which involves the evaluation of ranked variables via sequential backward elimination (SBE) and sequential forward selection (SFS). The implementation of CR-FS requires two main inputs, namely, start and stop number. The start number is the number of the highly ranked variables for the SBE while the stop number is the point at which the search for additional features during the SFS stage is halted. The setting of these critical parameters has always relied on trial and error which introduced subjectivity in the results obtained. The start and stop numbers are known to vary with each dataset. Drawing inspiration from overlapping coefficients, a method for comparing two probability density functions, empirical equations toward the estimation of start and stop number for a dataset were developed. All of the parameters in the empirical equations are obtained from the comparisons of the two probability density functions except the constant termed d. The equations were optimized using three real-world datasets. The optimum range of d was determined to be 0.48 to 0.57. An implementation of CR-FS using two new datasets demonstrated the validity of this approach. Partial least squares discriminant analysis (PLS-DA) model prediction accuracies increased from 90 and 96 to 100% for both datasets using start and stop numbers calculated with this approach. Additionally, there was a twofold increase in the explained variance captured in the first two principal components. Graphical abstract Here, we describe how to determine the start and stop numbers for an automated feature selection routine, ensuring that you get the best model you can for your data with minimal effort.
Keywords: Feature selection; Cluster resolution; Classification; Chemometrics; Overlapping coefficient; Fisher ratio

This work shows a novel analytical method for the simultaneous extraction of environmental emerging contaminants as benzenesulfonamides (BSAs), benzotriazoles (BTRs), and benzothiazoles (BTs) from water samples. Pristine multi-walled carbon nanotubes (MWCNTs), not yet tested for such analytes, are here employed as the sorbent phase for dispersive solid-phase extraction (d-SPE) followed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Quantitative sorption is gained by treating 50 mL sample with 100 mg MWCNTs (2 g L−1) in 10 min contact, both in tap and raw river water. After sorption, the analytes are quantitatively desorbed by microwaves (20 min, 160 °C, 250 W) by using 5 mL methanol-ethylacetate-acetic acid (10:70:20, v/v), according to the indications obtained by a chemometric study. The extract is reduced to small volume before analysis, thus reaching overall enrichment factors up to 400. Recovery of the entire procedure, evaluated on tap and surface water samples spiked with 0.1/0.5–50 μg L−1 of each analyte, was in the range 70–116%, with excellent inter-day precision (RSD < 7%). Selectivity and firm analyte identification were assured by MRM detection, and suitable sensitivity was obtained for determination of these pollutants in actual matrices (experimental MDLs 30–170 ng L−1). The proposed analytical method was applied to the analysis of surface water samples, containing concentrations of these contaminants ranging from 100 ng L−1 to 2 μg L−1. Pristine MWCNTs proved to be a valid alternative to other commercial sorbents, both in terms of cost and sorption capacity. Graphical abstract Determination of benzenesulfonamides, benzotriazoles, and benzothiazoles in environmental waters by dispersive multi-walled carbon nanotube extraction prior HPLC-MS
Keywords: Benzenesulfonamides; Benzothiazoles; Benzotriazoles; Carbon nanotubes; Microwaves; Solid-phase extraction

Erratum to: Exhaled breath online measurement for cervical cancer patients and healthy subjects by proton transfer reaction mass spectrometry by Wenzhao Zhou; Chaoqun Huang; Xue Zou; Yan Lu; Chengyin Shen; Xiping Ding; Hongzhi Wang; Haihe Jiang; Yannan Chu (6719-6719).