Analytical and Bioanalytical Chemistry (v.410, #13)

is a chemist by training and Professor for Effect-based Environmental Analysis at the Eberhard Karls Universität Tübingen. Since 2013 she is the speaker of the platform Environmental System Analysis at the university. Her research interests cover all aspects of analytical chemistry: instrumental development, analytical basic research, method development, and applications. A major focus is on electromigration separation techniques, including multidimensional separations and hyphenation to mass spectrometry. New methods are applied in various fields such as environmental (bio)analysis, forensics, clinical chemistry, and pharmacy with cooperation partners. Glyphosate is the world’s most heavily applied herbicide. Worldwide, re-approval processes by authorities are ongoing, accompanied by intense public and political discussions on its possible carcinogenic effects. Further aspects involve human dietary exposure, its fate in the environment, and its impact on ecosystems. Many of these aspects are not yet fully understood. In many instances, the analytical strategies developed and applied so far have strong limitations, given the challenging physicochemical characteristics of glyphosate and its metabolites. Analytical chemists are still faced with problems in method development, reachable precision, and detection limits. Thus, not all open research questions can be answered with current strategies. This feature article wishes to address further needs in glyphosate analysis to foster enhancement of analytical strategies.
Keywords: Pesticides; Ecotoxicology; Environment; AMPA; Monitoring; Speciation

Aptamer-facilitated mass cytometry by Gleb G. Mironov; Alexandre Bouzekri; Jessica Watson; Olga Loboda; Olga Ornatsky; Maxim V. Berezovski (3047-3051).
Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt’s lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstractBiotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.
Keywords: Mass cytometry; Flow cytometry; Aptamers; PTK7

The development of an easy to use, one-pot, environmentally friendly, non-invasive and label-free colorimetric probe for the determination of semen protamines, the biochemical marker of male fertility, using heparin gold nanoparticles (HAuNPs) is presented. The affinity of HAuNPs for protamines was due to the electrostatic interactions between polycationic protamine and polyanionic heparin. The binding of HAuNPs to protamine was characterized by variation in the plasmon absorption spectra followed by a visibly observable colour change of the solution from red to blue. We observed a red shift in the plasmon peak and the method exhibited linearity in the range of 10–70 ng/mL with a detection limit of 5 ng/mL, which is much lower than that reported for colorimetric sensors of protamine. The colour change and the variation in the absorbance of HAuNPs were highly specific for protamines in the presence of different interfering compounds and the method was successfully applied for determining protamine in real samples of semen and serum. Rather than a quantitative estimation, it seems that the method provides a quick screening between a large array of positive and negative samples and, moreover, it maintains the privacy of the user. The method appears to be simple and would be very useful in third-world countries where high-tech diagnostic aids are inaccessible to the majority of the population. Graphical AbstractHeparin gold nanoparticles aided visual detection of infertility
Keywords: Heparin; Gold nanoparticles; Protamines; Infertility; Surface plasmon absorption; Semen

Silicone wristbands compared with traditional polycyclic aromatic hydrocarbon exposure assessment methods by Holly M. Dixon; Richard P. Scott; Darrell Holmes; Lehyla Calero; Laurel D. Kincl; Katrina M. Waters; David E. Camann; Antonia M. Calafat; Julie B. Herbstman; Kim A. Anderson (3059-3071).
is a PhD candidate in toxicology at Oregon State University. Her research goals include characterizing personal chemical exposure and understanding how chemicals may influence human health, especially in communities facing environmental justice issues. is the manager of the Food Safety and Environmental Stewardship Laboratory at Oregon State University, as well as the senior chemist. He enjoys working with graduate students to push the boundaries of the science. is an associate professor of environmental and occupational health at Oregon State University. Her research focuses on controlling occupational and environmental exposures with the goal to reduce adverse health outcomes. is the division director for biological sciences research at the Pacific Northwest National Laboratory. Her research interests are focused on the integration of genomics, proteomics, metabolomics, and high-throughput screening data to enable the predictive mechanistic modeling of disease and toxicity pathways. is the chief of the Organic Analytical Toxicology Branch at the Division of Laboratory Sciences, National Center for Environmental Health of the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia. She currently leads CDC’s biomonitoring programs for assessing human exposure to pesticides; polycyclic aromatic hydrocarbons; flame retardants; persistent organic pollutants such as per- and polyfluoroalkyl substances; polybrominated diphenyl ethers; polychlorinated dibenzo-p-dioxins, furans, and biphenyls; and chemicals added to consumer and personal care products such as phthalates and phenols (e.g., bisphenol A, triclosan, parabens). is an associate professor in the Department of Environmental Health Sciences at the Columbia Mailman School of Public Health in New York City. She is a molecular and environmental epidemiologist at the Columbia Center for Children’s Environmental Health (CCCEH). At CCCEH, she directs two longitudinal birth cohort studies examining the effects of early-life environmental exposures—including polycyclic aromatic hydrocarbons, flame retardants, and phenols—on child health outcomes. is a professor in the Department of Environmental and Molecular Toxicology at Oregon State University, and an OSU Alumni Distinguished Professor. She directs the Food Safety and Environmental Stewardship Program at OSU. Since 2008, Dr. Anderson has been developing personal silicone wristband sampler technology for measuring an individual’s chemical exposures. Dr. Anderson is interested in developing personal chemical exposure surveys to better understand connections between chemical exposures, interventions, and health outcomes. Currently there is a lack of inexpensive, easy-to-use technology to evaluate human exposure to environmental chemicals, including polycyclic aromatic hydrocarbons (PAHs). This is the first study in which silicone wristbands were deployed alongside two traditional personal PAH exposure assessment methods: active air monitoring with samplers (i.e., polyurethane foam (PUF) and filter) housed in backpacks, and biological sampling with urine. We demonstrate that wristbands worn for 48 h in a non-occupational setting recover semivolatile PAHs, and we compare levels of PAHs in wristbands to PAHs in PUFs-filters and to hydroxy-PAH (OH-PAH) biomarkers in urine. We deployed all samplers simultaneously for 48 h on 22 pregnant women in an established urban birth cohort. Each woman provided one spot urine sample at the end of the 48-h period. Wristbands recovered PAHs with similar detection frequencies to PUFs-filters. Of the 62 PAHs tested for in the 22 wristbands, 51 PAHs were detected in at least one wristband. In this cohort of pregnant women, we found more significant correlations between OH-PAHs and PAHs in wristbands than between OH-PAHs and PAHs in PUFs-filters. Only two comparisons between PAHs in PUFs-filters and OH-PAHs correlated significantly (r s = 0.53 and p = 0.01; r s = 0.44 and p = 0.04), whereas six comparisons between PAHs in wristbands and OH-PAHs correlated significantly (r s = 0.44 to 0.76 and p = 0.04 to <0.0001). These results support the utility of wristbands as a biologically relevant exposure assessment tool which can be easily integrated into environmental health studies. Graphical abstractPAHs detected in samples collected from urban pregnant women
Keywords: Passive sampling; Active sampling; Biomonitoring; Personal monitoring; Environmental toxicology; Exposome

Geochemical wolframite fingerprinting – the likelihood ratio approach for laser ablation ICP-MS data by Agnieszka Martyna; Hans-Eike Gäbler; Andreas Bahr; Grzegorz Zadora (3073-3091).
is a doctor at the Department of Analytical Chemistry at the University of Silesia in Katowice in Poland. Her main interests include application of the statistical and chemometric tools for interpretation of the evidential value of physicochemical data for forensic purposes, with a special emphasis on the analysis of highly multivariate data. is an analytical chemist at the German Federal Institute for Geosciences and Natural Resources (BGR). His background is in inorganic analytical chemistry and the evaluation of the obtained data in the fields of mineral resources, hydrogeochemistry, soil chemistry, and disposal of radioactive waste. is a graduate engineer in analytical chemistry at the German Federal Institute for Geosciences and Natural Resources (BGR). He is an expert in the development of database applications and the processing of geochemical data with focus on applied statistics. is an associated professor at the Institute of Forensic Research in Krakow in Poland. He also holds a position at the Department of Analytical Chemistry at the University of Silesia in Katowice. He is a forensic expert in the field of physicochemical analysis of microtraces and blood pattern analysis and he mainly focuses on the development of the statistical tools assisting in data interpretation. Wolframite has been specified as a ‘conflict mineral’ by a U.S. Government Act, which obliges companies that use these minerals to report their origin. Minerals originating from conflict regions in the Democratic Republic of the Congo shall be excluded from the market as their illegal mining, trading, and taxation are supposed to fuel ongoing violent conflicts. The German Federal Institute for Geosciences and Natural Resources (BGR) developed a geochemical fingerprinting method for wolframite based on laser ablation inductively coupled plasma-mass spectrometry. Concentrations of 46 elements in about 5300 wolframite grains from 64 mines were determined. The issue of verifying the declared origins of the wolframite samples may be framed as a forensic problem by considering two contrasting hypotheses: the examined sample and a sample collected from the declared mine originate from the same mine (H1), and the two samples come from different mines (H2). The solution is found using the likelihood ratio (LR) theory. On account of the multidimensionality, the lack of normal distribution of data within each sample, and the huge within-sample dispersion in relation to the dispersion between samples, the classic LR models had to be modified. Robust principal component analysis and linear discriminant analysis were used to characterize samples. The similarity of two samples was expressed by Kolmogorov-Smirnov distances, which were interpreted in view of H1 and H2 hypotheses within the LR framework. The performance of the models, controlled by the levels of incorrect responses and the empirical cross entropy, demonstrated that the proposed LR models are successful in verifying the authenticity of the wolframite samples. Graphical abstractGeochemical wolframite fingerprinting
Keywords: Wolframite; Fingerprinting; Laser ablation ICP-MS; Likelihood ratio approach; Chemometrics

In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine–Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine–Zn(II)–pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis. Graphical abstractA simple and label-free fluorescence assay for SNP detection by coupling BRCA with selective fluorescence detection of pyrophosphate using the terpyridine–Zn(II) complex.
Keywords: Single-nucleotide polymorphism; Branched rolling circle amplification reaction; Terpyridine–Zn(II); Fluorometry

Development of quantitative screen for 1550 chemicals with GC-MS by Alan J. Bergmann; Gary L. Points; Richard P. Scott; Glenn Wilson; Kim A. Anderson (3101-3110).
With hundreds of thousands of chemicals in the environment, effective monitoring requires high-throughput analytical techniques. This paper presents a quantitative screening method for 1550 chemicals based on statistical modeling of responses with identification and integration performed using deconvolution reporting software. The method was evaluated with representative environmental samples. We tested biological extracts, low-density polyethylene, and silicone passive sampling devices spiked with known concentrations of 196 representative chemicals. A multiple linear regression (R 2 = 0.80) was developed with molecular weight, logP, polar surface area, and fractional ion abundance to predict chemical responses within a factor of 2.5. Linearity beyond the calibration had R 2 > 0.97 for three orders of magnitude. Median limits of quantitation were estimated to be 201 pg/μL (1.9× standard deviation). The number of detected chemicals and the accuracy of quantitation were similar for environmental samples and standard solutions. To our knowledge, this is the most precise method for the largest number of semi-volatile organic chemicals lacking authentic standards. Accessible instrumentation and software make this method cost effective in quantifying a large, customizable list of chemicals. When paired with silicone wristband passive samplers, this quantitative screen will be very useful for epidemiology where binning of concentrations is common. Graphical abstractA multiple linear regression of chemical responses measured with GC-MS allowed quantitation of 1550 chemicals in samples such as silicone wristbands.
Keywords: Gas chromatography; Multiple linear regression; Chemometrics; Response prediction; Automated mass spectral deconvolution and identification system (AMDIS); Passive sampling devices

Determination of lysine content based on an in situ pretreatment and headspace gas chromatographic measurement technique by Xiao-fang Wan; Bao-lian Liu; Teng Yu; Ning Yan; Xin-Sheng Chai; You-ming Li; Guang-xue Chen (3111-3117).
This work reports on a simple method for the determination of lysine content by an in situ sample pretreatment and headspace gas chromatographic measurement (HS-GC) technique, based on carbon dioxide (CO2) formation from the pretreatment reaction (between lysine and ninhydrin solution) in a closed vial. It was observed that complete lysine conversion to CO2 could be achieved within 60 min at 60 °C in a phosphate buffer medium (pH = 4.0), with a minimum molar ratio of ninhydrin/lysine of 16. The results showed that the method had a good precision (RSD < 5.23%) and accuracy (within 6.80%), compared to the results measured by a reference method (ninhydrin spectroscopic method). Due to the feature of in situ sample pretreatment and headspace measurement, the present method becomes very simple and particularly suitable to be used for batch sample analysis in lysine-related research and applications. Graphical abstractThe flow path of the reaction and HS-GC measurement for the lysine analysis
Keywords: Lysine; Headspace; Gas chromatography; Ninhydrin; Nanofibrillated cellulose

Plasma membrane (PM) and mitochondrial membrane (MM) proteins of rat hippocampal neurons were identified and comparatively characterized on the basis of a sequential digestion-centered combinative strategy for sample treatment. A total of 478 membrane proteins were identified, of which 240 had PM localization, 170 had MM localization, and 33 had both of the two subcellular localizations. Compared with the PM proteome, the MM proteome not only was smaller, more basic, and more hydrophobic, but also had a narrower protein molecular mass distribution range and a higher proportion of transmembrane proteins. By functional enrichment analysis, 287 molecular function terms for the PM proteome and 173 for the MM proteome were obtained. The MM proteome had a lower percentage of binding function terms and a higher percentage of catalysis function terms than the PM proteome, suggesting that mitochondrial proteins were more inclined to affect the physiological and biochemical processes by binding various molecules and as enzymes. Biological process enrichment showed that the genes of the PM and MM proteomes were mapped to 1104 and 460 biological processes, respectively. The biological processes with the most mapped genes of the PM proteome included those involved in vesicle recycling, transmitter release, neuronal development, protein and ion transport, etc., whereas those involved in electron transport, ATP synthesis, mitochondrial transport, mitochondrial apoptosis, etc., were the most gene-mapped biological processes for the MM proteome. The present work has deepened our understanding of the structure and function of hippocampal neurons and provided reference methods for research in the related field. Graphical abstractFunctional comparison of the plasma membrane and mitochondrial membrane proteomes
Keywords: Subcellular proteome; Plasma membrane; Mitochondrial membrane; Comparative characterization; Combinative strategy

Novel molecularly imprinted polymers (MIPs) based on denatured casein nanoparticle (DCP)-stabilized Pickering emulsions were developed for the first time. Casein, a phosphoprotein, is the main protein in milk. In this work, DCPs were solely used as Pickering-type interfacial emulsifiers for fabrication of MIPs for the selective recognition of proteins for the first time. DCPs were prepared by acidification and heat denaturation (at 80 °C) of casein. Their dispersions have satisfactory colloidal stability over a wide pH range. The DCPs acted as natural, food-grade, and edible interfacial emulsifiers, and adsorbed at the oil–water interface to form Pickering emulsions. After the polymerization of monomers, the template protein was removed by elution. During the elution, the interfacial DCPs were also removed, allowing more imprinted cavities to become exposed. The interfacial imprinting technology causes nearly all the imprinted sites to locate on the surface of the polymeric material. Therefore, the MIPs obtained exhibit fast rebinding and excellent specific recognition ability toward the analytes. Overall, this work provides a promising method for designing and fabricating natural-protein-based structured emulsions to prepare MIPs and thus offers new insight into protein separation and purification. Graphical AbstractPickering emulsions stabilized by denatured casein particles.
Keywords: Molecularly imprinted polymers; Pickering emulsion; Food grade; Casein; Protein separation

Polyethylene glycol 300 is commonly used as a base material for “analyte protection” in multiresidue pesticide analysis via gas chromatography-mass spectrometry. However, the disadvantage of the co-injection method using polyethylene glycol 300 is that it causes peak instability in α-cyano pyrethroids (type II pyrethroids) such as fluvalinate. In this study, we confirmed the instability phenomenon in type II pyrethroids and developed novel analyte protectants for acetone/n-hexane mixture solution to suppress the phenomenon. Our findings revealed that among the examined additive compounds, three lipophilic ascorbic acid derivatives, 3-O-ethyl-L-ascorbic acid, 6-O-palmitoyl-L-ascorbic acid, and 6-O-stearoyl-L-ascorbic acid, could effectively stabilize the type II pyrethroids in the presence of polyethylene glycol 300. A mixture of the three ascorbic acid derivatives and polyethylene glycol 300 proved to be an effective analyte protectant for multiresidue pesticide analysis. Further, we designed and evaluated a new combination of analyte protectant compounds without using polyethylene glycol or the troublesome hydrophilic compounds. Consequently, we obtained a set of 10 medium- and long-chain saturated fatty acids as an effective analyte protectant suitable for acetone/n-hexane solution that did not cause peak instability in type II pyrethroids. These analyte protectants will be useful in multiresidue pesticide analysis by gas chromatography-mass spectrometry in terms of ruggedness and reliable quantitativeness. Graphical abstractComparison of effectiveness of the addition of lipophilic derivatives of ascorbic acid in controlling the instability phenomenon of fluvalinate with polyethylene glycol 300
Keywords: Analyte protectant; Pesticide residue analysis; α-Cyano pyrethroid; Type II pyrethroid

Highly sensitive detection of a small molecule by a paired labels recognition system based lateral flow assay by Leina Dou; Bingxin Zhao; Tong Bu; Wentao Zhang; Qiong Huang; Lingzhi Yan; Lunjie Huang; Yanru Wang; Jianlong Wang; Daohong Zhang (3161-3170).
Small molecules are difficult to detect by conventional gold lateral flow assay (GLFA) sensitively because the test system must satisfy the conflict requirements between enough signal intensity and limited antibody (Ab) amount. In this work, a paired labels recognition (PLR)-based biosensor was designed by utilizing the specific binding of Ab and secondary antibody (anti-Ab) to enhance signal intensity and reduce antibody amount applied in small molecule detection. The PLR amplification system is fabricated by self-assembling the common detection probe, Au-labeled Ab (Au-Ab), and the signal booster, Au-labeled anti-Ab (Au-anti-Ab). Benefiting from this, a powerful network structure can be generated to accumulate numerous gold nanoparticles (GNPs) and thus significantly strengthen the signal intensity of detection. Therefore, a lower Ab amount will be applied to offer adequate signal strength, and further, the limit of detection will be obviously downregulated due to the more effective competition reaction. Using furazolidone (FZD) as a model analyte, we achieve a detection limit of as low as 1 ng mL−1, which was at least fivefold improved over that of the traditional GLFA. Furthermore, the practicality of this strategy was certificated in five different food samples. Graphical abstractA paired labels recognition (PLR) amplification system is fabricated by self-assembling the common detection probe, Au-labeled Ab (Au-Ab), and the signal booster, Au-labeled anti-Ab (Au-anti-Ab). In this novel strategy, owing to the recognition of both Ab and anti-Ab labeled on gold nanoparticles (GNPs), a powerful network structure can be generated to accumulate numerous GNPs and thus significantly strengthen the signal intensity of detection.
Keywords: Lateral flow assay; Gold nanoparticles; Signal amplification; Furazolidone; Food analysis

Analytical procedure for the determination of very volatile organic compounds (C3–C6) in indoor air by Alexandra Schieweck; Jan Gunschera; Deniz Varol; Tunga Salthammer (3171-3183).
The substance group of very volatile organic compounds (VVOCs) is moving into the focus of indoor air analysis, facing ongoing regulations at international and European levels targeting on indoor air quality and human health. However, there exists at present no validated analysis for the identification and quantification of VVOCs in indoor air. Therefore, the present study targeted on the development of an analytical method in order to sample the maximum possible quantity of VVOCs in indoor air on solid sorbents with subsequent analysis by thermal desorption and coupled gas chromatography/mass spectrometry (TDS-GC/MS). For this purpose, it was necessary to investigate the performance of available sorbents and to optimize the parameters of GC/MS analysis. Stainless steel tubes filled with Carbograph 5TD were applied successfully for low-volume sampling (2–4 l) with minimal breakthrough (< 1%). With the developed method, VVOCs between C3 and C6 of different volatility and polarity  can be detected even in trace quantities with low limits of quantitation (LOQ; 1–3 μg m−3). Limitations occur for low molecular weight compounds ≤C3, especially for polar substances, such as carboxylic acids and for some aldehydes and alcohols. Consequently, established methods for the quantification of these compounds in indoor air cannot be fully substituted yet. At least three different analytical techniques are needed to cover the large spectrum of relevant VVOCs in indoor air. In addition, unexpected reaction products might occur and need to be taken into account to avoid misinterpretation of chromatographic signals. Graphical abstractSolid sorbent sampling of VVOCs (C3-C6) in indoor air with subsequent TDS-GC/MS analysis
Keywords: VVOC; Indoor air; Analysis; Gas chromatography; Mass spectrometry; Thermal desorption

Frankincense gum resin secreted from Boswellia papyrifera was analysed by comprehensive 2D gas chromatography hyphenated with accurate mass time-of-flight mass spectrometry (GC×GC−accTOFMS). Direct multiple injection experiments with stepwise isothermal temperature programming were then performed to construct isovolatility curves for reference alkane series in GC×GC. This provides access to calculation of second dimensional retention indices (2 I). More than 500 peaks were detected and 220 compounds mainly comprising monoterpenes, sesquiterpenes, diterpenes and oxygenated forms of these compounds were identified according to their 1 I, 2 I and accurate mass data. The study demonstrates the capability of GC×GC−accTOFMS with retention data on two separate column phases, as an approach for improved component identification. A greater number of identified and/or tentatively identified terpenoids in this traditional Chinese medicine allow for a more comprehensive coverage of the volatile composition of frankincense.
Keywords: Comprehensive 2D gas chromatography; 2D index calculation; Exact mass TOFMS; Isovolatility; Second dimension retention index; 2D retention structure

Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control by Simone Albrecht; Christian Kaisermayer; David Reinhart; Monica Ambrose; Renate Kunert; Anna Lindeberg; Jonathan Bones (3197-3207).
The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MSE was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 106 dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstractOverview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death
Keywords: Biomanufacturing; Cell death; CHO; Proteomics; LC-MRM-MS; Targeted proteomics

Fluorescence turn-on detection of target sequence DNA based on silicon nanodot-mediated quenching by Yanan Zhang; Xinping Ning; Guobin Mao; Xinghu Ji; Zhike He (3209-3216).
We have developed a new enzyme-free method for target sequence DNA detection based on the dynamic quenching of fluorescent silicon nanodots (SiNDs) toward Cy5-tagged DNA probe. Fascinatingly, the water-soluble SiNDs can quench the fluorescence of cyanine (Cy5) in Cy5-tagged DNA probe in homogeneous solution, and the fluorescence of Cy5-tagged DNA probe can be restored in the presence of target sequence DNA (the synthetic target miRNA-27a). Based on this phenomenon, a SiND-featured fluorescent sensor has been constructed for “turn-on” detection of the synthetic target miRNA-27a for the first time. This newly developed approach possesses the merits of low cost, simple design, and convenient operation since no enzymatic reaction, toxic reagents, or separation procedures are involved. The established method achieves a detection limit of 0.16 nM, and the relative standard deviation of this method is 9% (1 nM, n = 5). The linear range is 0.5–20 nM, and the recoveries in spiked human fluids are in the range of 90–122%. This protocol provides a new tactic in the development of the nonenzymic miRNA biosensors and opens a promising avenue for early diagnosis of miRNA-associated disease. Graphical abstractThe SiND-based fluorescent sensor for detection of S-miR-27a
Keywords: Silicon nanodots; DNA probe; Fluorescent sensor; The synthetic target miRNA-27a