Analytical and Bioanalytical Chemistry (v.408, #19)

Single-particle-ICP-MS advances by Antonio R. Montoro Bustos; Michael R. Winchester (5051-5052).
is a Guest Scientist at the National Institute of Standards and Technology (NIST) in Gaithersburg, Maryland (USA). He received his Ph.D. degree in Analytical Chemistry from The University of Oviedo (Spain) under the direction of Dr. Jorge Ruiz Encinar and Prof. Alfredo Sanz - Medel. His research efforts are focused on the application of single-particle ICP-MS for the characterization of nanomaterials at environmentally relevant concentrations, including the validation of the technique, the evaluation of the role of different coatings on the stability of nanoparticles and the study of matrix effects on spICP-MS analytical performance. is a Supervisory Research Chemist and Leader of the Inorganic Measurement Science Group in the Chemical Sciences Division of NIST in Gaithersburg, Maryland (USA). He began his career at NIST in 1991 as a National Research Council Postdoctoral Associate, immediately after earning the Ph.D. degree in Analytical Chemistry from Clemson University under the direction of Prof. R. Kenneth Marcus. Over the years, his research interests have involved glow discharge spectroscopy; Fourier transform spectroscopy in the ultraviolet/visible spectral range; ultralow bias, small uncertainty analysis using atomic spectroscopy; advancements in reference materials; and other topics. His most recent research interest is in the area of physicochemical characterizations of engineered nanomaterials. The NIST group that he leads has dedicated research programs in this area, as well as in seawater pH metrology and other areas, and is also responsible for hundreds of NIST reference materials.

Single Particle ICP-MS: Advances toward routine analysis of nanomaterials by Manuel D. Montaño; John W. Olesik; Angela G. Barber; Katie Challis; James F. Ranville (5053-5074).
From its early beginnings in characterizing aerosol particles to its recent applications for investigating natural waters and waste streams, single particle inductively coupled plasma-mass spectrometry (spICP-MS) has proven to be a powerful technique for the detection and characterization of aqueous dispersions of metal-containing nanomaterials. Combining the high-throughput of an ensemble technique with the specificity of a single particle counting technique and the elemental specificity of ICP-MS, spICP-MS is capable of rapidly providing researchers with information pertaining to size, size distribution, particle number concentration, and major elemental composition with minimal sample perturbation. Recently, advances in data acquisition, signal processing, and the implementation of alternative mass analyzers (e.g., time-of-flight) has resulted in a wider breadth of particle analyses and made significant progress toward overcoming many of the challenges in the quantitative analysis of nanoparticles. This review provides an overview of spICP-MS development from a niche technique to application for routine analysis, a discussion of the key issues for quantitative analysis, and examples of its further advancement for analysis of increasingly complex environmental and biological samples. Graphical Abstract Single particle ICP-MS workflow for the analysis of suspended nanoparticles
Keywords: Single particle ICP-MS; ICP-TOF-MS; Engineered nanoparticles; Environmental analysis; Core-shell nanoparticles; FFF-ICP-MS

Drift correction of the dissolved signal in single particle ICPMS by Geert Cornelis; Sebastien Rauch (5075-5087).
A method is presented where drift, the random fluctuation of the signal intensity, is compensated for based on the estimation of the drift function by a moving average. It was shown using single particle ICPMS (spICPMS) measurements of 10 and 60 nm Au NPs that drift reduces accuracy of spICPMS analysis at the calibration stage and during calculations of the particle size distribution (PSD), but that the present method can again correct the average signal intensity as well as the signal distribution of particle-containing samples skewed by drift. Moreover, deconvolution, a method that models signal distributions of dissolved signals, fails in some cases when using standards and samples affected by drift, but the present method was shown to improve accuracy again. Relatively high particle signals have to be removed prior to drift correction in this procedure, which was done using a 3 × sigma method, and the signals are treated separately and added again. The method can also correct for flicker noise that increases when signal intensity is increased because of drift. The accuracy was improved in many cases when flicker correction was used, but when accurate results were obtained despite drift, the correction procedures did not reduce accuracy. The procedure may be useful to extract results from experimental runs that would otherwise have to be run again. Graphical Abstract A method is presented where a spICP-MS signal affected by drift (left) is corrected (right) by adjusting the local (moving) averages (green) and standard deviations (purple) to the respective values at a reference time (red). In combination with removing particle events (blue) in the case of calibration standards, this method is shown to obtain particle size distributions where that would otherwise be impossible, even when the deconvolution method is used to discriminate dissolved and particle signals
Keywords: Nanoparticles/nanotechnology; Mass spectrometry/ICPMS; Chemometrics/statistics; Metals/heavy metals

Evaluation of number concentration quantification by single-particle inductively coupled plasma mass spectrometry: microsecond vs. millisecond dwell times by Isabel Abad-Álvaro; Elena Peña-Vázquez; Eduardo Bolea; Pilar Bermejo-Barrera; Juan R. Castillo; Francisco Laborda (5089-5097).
The quality of the quantitative information in single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) depends directly on the number concentration of the nanoparticles in the sample analyzed, which is proportional to the flux of nanoparticles through the plasma. Particle number concentrations must be selected in accordance with the data acquisition frequency, to control the precision from counting statistics and the bias, which is produced by the occurrence of multiple-particle events recorded as single-particle events. With quadrupole mass spectrometers, the frequency of data acquisition is directly controlled by the dwell time. The effect of dwell times from milli- to microseconds (10 ms, 5 ms, 100 μs, and 50 μs) on the quality of the quantitative data has been studied. Working with dwell times in the millisecond range, precision figures about 5 % were achieved, whereas using microsecond dwell times, the suitable fluxes of nanoparticles are higher and precision was reduced down to 1 %; this was independent of the dwell time selected. Moreover, due to the lower occurrence of multiple-nanoparticle events, linear ranges are wider when dwell times equal to or shorter than 100 μs are used. A calculation tool is provided to determine the optimal concentration for any instrument or experimental conditions selected. On the other hand, the use of dwell times in the microsecond range reduces significantly the contribution of the background and/or the presence of dissolved species, in comparison with the use of millisecond dwell times. Although the use of dwell times equal to or shorter than 100 μs offers improved performance working in single-particle mode, the use of conventional dwell times (3–10 ms) should not be discarded, once their limitations are known.
Keywords: Single particle; SP-ICP-MS; Nanoparticle; Quantification; Number concentration

The effect of ICP-MS instrument sensitivity drift on the accuracy of nanoparticle (NP) size measurements using single particle (sp)ICP-MS is investigated. Theoretical modeling and experimental measurements of the impact of instrument sensitivity drift are in agreement and indicate that drift can impact the measured size of spherical NPs by up to 25 %. Given this substantial bias in the measured size, a method was developed using an internal standard to correct for the impact of drift and was shown to accurately correct for a decrease in instrument sensitivity of up to 50 % for 30 and 60 nm gold nanoparticles. Graphical Abstract Correction of nanoparticle size measurement by spICP-MS using an internal standard
Keywords: Drift correction; Gold nanoparticles; Instrument sensitivity drift; Internal standard; Single-particle ICP-MS

Hydrodynamic chromatography coupled to single-particle ICP-MS for the simultaneous characterization of AgNPs and determination of dissolved Ag in plasma and blood of burn patients by Marco Roman; Chiara Rigo; Hiram Castillo-Michel; Ivan Munivrana; Vincenzo Vindigni; Ivan Mičetić; Federico Benetti; Laura Manodori; Warren R. L. Cairns (5109-5124).
Silver nanoparticles (AgNPs) are increasingly used in medical devices as innovative antibacterial agents, but no data are currently available on their chemical transformations and fate in vivo in the human body, particularly on their potential to reach the circulatory system. To study the processes involving AgNPs in human plasma and blood, we developed an analytical method based on hydrodynamic chromatography (HDC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) in single-particle detection mode. An innovative algorithm was implemented to deconvolute the signals of dissolved Ag and AgNPs and to extrapolate a multiparametric characterization of the particles in the same chromatogram. From a single injection, the method provides the concentration of dissolved Ag and the distribution of AgNPs in terms of hydrodynamic diameter, mass-derived diameter, number and mass concentration. This analytical approach is robust and suitable to study quantitatively the dynamics and kinetics of AgNPs in complex biological fluids, including processes such as agglomeration, dissolution and formation of protein coronas. The method was applied to study the transformations of AgNP standards and an AgNP-coated dressing in human plasma, supported by micro X-ray fluorescence (μXRF) and micro X-ray absorption near-edge spectroscopy (μXANES) speciation analysis and imaging, and to investigate, for the first time, the possible presence of AgNPs in the blood of three burn patients treated with the same dressing. Together with our previous studies, the results strongly support the hypothesis that the systemic mobilization of the metal after topical administration of AgNPs is driven by their dissolution in situ. Graphical Abstract Simplified scheme of the combined analytical approach adopted for studying the chemical dynamics of AgNPs in human plasma/blood
Keywords: Silver nanoparticles; Hydrodynamic chromatography; Single-particle ICP-MS; Synchrotron radiation; Burns; Wound dressings

Combining single-particle inductively coupled plasma mass spectrometry and X-ray absorption spectroscopy to evaluate the release of colloidal arsenic from environmental samples by Miguel Angel Gomez-Gonzalez; Eduardo Bolea; Peggy A. O’Day; Javier Garcia-Guinea; Fernando Garrido; Francisco Laborda (5125-5135).
Detection and sizing of natural colloids involved in the release and transport of toxic metals and metalloids is essential to understand and model their environmental effects. Single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) was applied for the detection of arsenic-bearing particles released from mine wastes. Arsenic-bearing particles were detected in leachates from mine wastes, with a mass-per-particle detection limit of 0.64 ng of arsenic. Conversion of the mass-per-particle information provided by SP-ICP-MS into size information requires knowledge of the nature of the particles; therefore, synchrotron-based X-ray absorption spectroscopy (XAS) was used to identify scorodite (FeAsO4·2H2O) as the main species in the colloidal particles isolated by ultrafiltration. The size of the scorodite particles detected in the leachates was below 300–350 nm, in good agreement with the values obtained by TEM. The size of the particles detected by SP-ICP-MS was determined as the average edge of scorodite crystals, which show a rhombic dipyramidal form, achieving a size detection limit of 117 nm. The combined use of SP-ICP-MS and XAS allowed detection, identification, and size determination of scorodite particles released from mine wastes, suggesting their potential to transport arsenic. Graphical abstract Analytical approach for the detection and size characterization of As-bearing particles by SP-ICP-MS and XAS in environmental samples
Keywords: Single particle; SP-ICP-MS; XAS; Arsenic; Colloids; Scorodite

Detection of zinc oxide and cerium dioxide nanoparticles during drinking water treatment by rapid single particle ICP-MS methods by Ariel R. Donovan; Craig D. Adams; Yinfa Ma; Chady Stephan; Todd Eichholz; Honglan Shi (5137-5145).
Nanoparticles (NPs) entering water systems are an emerging concern as NPs are more frequently manufactured and used. Single particle inductively coupled plasma-mass spectrometry (SP-ICP-MS) methods were validated to detect Zn- and Ce-containing NPs in surface and drinking water using a short dwell time of 0.1 ms or lower, ensuring precision in single particle detection while eliminating the need for sample preparation. Using this technique, information regarding NP size, size distribution, particle concentration, and dissolved ion concentrations was obtained simultaneously. The fates of Zn- and Ce-NPs, including those found in river water and added engineered NPs, were evaluated by simulating a typical drinking water treatment process. Lime softening, alum coagulation, powdered activated carbon sorption, and disinfection by free chlorine were simulated sequentially using river water. Lime softening removed 38–53 % of Zn-containing and ZnO NPs and >99 % of Ce-containing and CeO2 NPs. Zn-containing and ZnO NP removal increased to 61–74 % and 77–79 % after alum coagulation and disinfection, respectively. Source and drinking water samples were collected from three large drinking water treatment facilities and analyzed for Zn- and Ce-containing NPs. Each facility had these types of NPs present. In all cases, particle concentrations were reduced by a minimum of 60 % and most were reduced by >95 % from source water to finished drinking water. This study concludes that uncoated ZnO and CeO2 NPs may be effectively removed by conventional drinking water treatments including lime softening and alum coagulation.
Keywords: Single particle ICP-MS; ZnO and CeO2 nanoparticles; Nanoparticle occurrence and removal; Nanoparticle characterization; Drinking water treatment

Engineered nanoparticles (ENP) are increasingly being incorporated into consumer products and reaching the environment at a growing rate. Unfortunately, few analytical techniques are available that allow the detection of ENP in complex environmental matrices. The major limitations with existing techniques are their relatively high detection limits and their inability to distinguish ENP from other chemical forms (e.g. ions, dissolved) or from natural colloids. Of the matrices that are considered to be a priority for method development, ENP are predicted to be found at relatively high concentrations in wastewaters and wastewater biosolids. In this paper, we demonstrate the capability of hydrodynamic chromatography (HDC) coupled to inductively coupled plasma mass spectrometry (ICPMS), in its classical and single particle modes (SP ICPMS), to identify ENP in wastewater influents and effluents. The paper first focuses on the detection of standard silver nanoparticles (Ag NP) and their mixtures, showing that significant dissolution of the Ag NP was likely to occur. For the Ag NP, detection limits of 0.03 μg L−1 were found for the HDC ICPMS whereas 0.1 μg L−1 was determined for the HDC SP ICPMS (based on results for the 80 nm Ag NP). In the second part of the paper, HDC ICPMS and HDC SP ICPMS were performed on some unspiked natural samples (wastewaters, river water). While nanosilver was below detection limits, it was possible to identify some (likely natural) Cu nanoparticles using the developed separation technology.
Keywords: Nanosilver; Nanoparticles; Hydrodynamic chromatography; Single particle ICPMS; Wastewaters

Single particle ICP-MS method development for the determination of plant uptake and accumulation of CeO2 nanoparticles by Yongbo Dan; Xingmao Ma; Weilan Zhang; Kun Liu; Chady Stephan; Honglan Shi (5157-5167).
Cerium dioxide nanoparticles (CeO2NPs) are among the most broadly used engineered nanoparticles that will be increasingly released into the environment. Thus, understanding their uptake, transportation, and transformation in plants, especially food crops, is critical because it represents a potential pathway for human consumption. One of the primary challenges for the endeavor is the inadequacy of current analytical methodologies to characterize and quantify the nanomaterial in complex biological samples at environmentally relevant concentrations. Herein, a method was developed using single particle-inductively coupled plasma-mass spectrometry (SP-ICP-MS) technology to simultaneously detect the size and size distribution of particulate Ce, particle concentration, and dissolved cerium in the shoots of four plant species including cucumber, tomato, soybean, and pumpkin. An enzymatic digestion method with Macerozyme R-10 enzyme previously used for gold nanoparticle extraction from the tomato plant was adapted successfully for CeO2NP extraction from all four plant species. This study is the first to report and demonstrate the presence of dissolved cerium in plant seedling shoots exposed to CeO2NPs hydroponically. The extent of plant uptake and accumulation appears to be dependent on the plant species, requiring further systematic investigation of the mechanisms.
Keywords: CeO2 nanoparticles; Single particle ICP-MS; Plant uptake of nanoparticles; Enzymatic digestion; Nanoparticle characterization; Biotransformation

Single particle ICP-MS as a tool for determining the stability of silver nanoparticles in aquatic matrixes under various environmental conditions, including treatment by ozonation by Lena Telgmann; Michael Thanh Khoa Nguyen; Li Shen; Viviane Yargeau; Holger Hintelmann; Chris D. Metcalfe (5169-5177).
Silver nanoparticles (AgNPs) are used in a large number of consumer products due to their antimicrobial and antifungal properties, and these materials may be discharged into municipal wastewater. Wastewater treatment, including advanced oxidation processes (AOPs), may modify the forms of silver in wastewater before they are discharged into surface waters. In addition, little is known about the changes in AgNPs that occur in natural waters under different environmental conditions. In this project, we utilized single particle ICP-MS (spICP-MS) and dynamic light scattering (DLS) analytical techniques to evaluate changes in the number and size of AgNPs in laboratory experiments with milliQ water under different environmental conditions, as well as during ozonation. Changes in the number and size of AgNPs determined by spICP-MS were evidence of altered stability of the nanoparticles. Increased rates of dissolution occurred under extremes of pH. Lower temperature decreased the rate of dissolution of AgNP relative to the dissolution in treatments at room temperature. The addition of chloride resulted in the loss of AgNPs from suspension due to agglomeration and precipitation. Ozonation led to a rapid decline in the number and size of AgNPs, as indicated by both spICP-MS and DLS analysis. An increase in the concentration of dissolved silver in the ozone treatments was evidence that changes in particle size were a result of oxidative dissolution of AgNPs to silver ion. Graphical abstract Single particle ICP-MS is used to evaluate dissolution of silver nanoparticles under different environmental conditions, including water treatment by ozonation
Keywords: Single particle ICP-MS; Dynamic light scattering; Nanoparticles/nanotechnology; Silver; Ozonation

The recent development of a homogeneous time-resolved Förster resonance energy transfer (TR–FRET) immunoassay enables one-step, rapid (minutes), and direct detection compared to the multistep, time-consuming (hours), heterogeneous ELISA-type immunoassays. The use of the time-resolved effect of a donor lanthanide complex with a delay time of microseconds and large Stokes shift enables the separation of positive signals from the background autofluorescence of the sample. However, this study shows that the sample matrices directly interfere with donor fluorescence and that interference cannot be eliminated by time-resolved settings alone. Moreover, the reduction in donor emission did not appear to be equivalent to the reduction in acceptor emission, resulting in incorrect FRET signal measurements. To overcome this limitation, an internal standard approach was developed using an isotype control antibody. This new approach was used to develop TR–FRET assays for rapid detection (15–30 min) of Bacillus anthracis spores and botulinum toxin (type E) in beverages, which are major concerns in bioterrorism involving deliberate food contamination. Additionally, we demonstrate the detection of B. anthracis-secreted protective antigen (PA) and the Yersinia pestis-secreted markers F1 and LcrV in blood cultures, which are early markers of bacteremia in infected hosts following a possible bioterror attack. The use of an internal standard enables the calculation of correct ΔF values without the need for an external standard. Thus, the use of the internal standard approach in homogeneous immunoassays facilitates the examination of any sample regardless of its origin, and therefore expands the applicability of TR–FRET assays for complex matrices.
Keywords: Time-resolved fluorescence; FRET; Yersinia pestis ; Bacillus anthracis ; Botulinum toxin

Rapid detection of hazardous chemicals in textiles by direct analysis in real-time mass spectrometry (DART-MS) by Borbála Antal; Ákos Kuki; Lajos Nagy; Tibor Nagy; Miklós Zsuga; Sándor Kéki (5189-5198).
Residues of chemicals on clothing products were examined by direct analysis in real-time (DART) mass spectrometry. Our experiments have revealed the presence of more than 40 chemicals in 15 different clothing items. The identification was confirmed by DART tandem mass spectrometry (MS/MS) experiments for 14 compounds. The most commonly detected hazardous substances were nonylphenol ethoxylates (NPEs), phthalic acid esters (phthalates), amines released by azo dyes, and quinoline derivates. DART-MS was able to detect NPEs on the skin of the person wearing the clothing item contaminated by NPE residuals. Automated data acquisition and processing method was developed and tested for the recognition of NPE residues thereby reducing the analysis time.
Keywords: Direct analysis in real-time tandem mass spectrometry (DART-MS; DART-MS/MS); Hazardous substances; Textile articles; Nonylphenol ethoxylate (NPE); Phthalic acid ester (phthalate); Quinolines

Mesoporous carbon-containing voltammetric biosensor for determination of tyramine in food products by Jolanta Kochana; Karolina Wapiennik; Paweł Knihnicki; Aleksandra Pollap; Paula Janus; Marcin Oszajca; Piotr Kuśtrowski (5199-5210).
A voltammetric biosensor based on tyrosinase (TYR) was developed for determination of tyramine. Carbon material (multi-walled carbon nanotubes or mesoporous carbon CMK-3-type), polycationic polymer—i.e., poly(diallyldimethylammonium chloride) (PDDA), and Nafion were incorporated into titania dioxide sol (TiO2) to create an immobilization matrix. The features of the formed matrix were studied by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The analytical performance of the developed biosensor was evaluated with respect to linear range, sensitivity, limit of detection, long-term stability, repeatability, and reproducibility. The biosensor exhibited electrocatalytic activity toward tyramine oxidation within a linear range from 6 to 130 μM, high sensitivity of 486 μA mM−1 cm−2, and limit of detection of 1.5 μM. The apparent Michaelis–Menten constant was calculated to be 66.0 μM indicating a high biological affinity of the developed biosensor for tyramine. Furthermore, its usefulness in determination of tyramine in food product samples was also verified. Graphical abstract Different food samples were analyzed to determine tyramine using biosensor based on tyrosinase
Keywords: Biosensor; Tyramine; Food; Mesoporous carbon; Tyrosinase; Voltammetry

In this work, polyethyleneimine grafted silica-coated nanoscale zero valent iron (Fe@SiO2@PEI) has been successfully synthesized and was investigated to be an effective adsorbent for efficient enrichment of five phthalate esters such as diphenyl phthalate, dibenzyl phthalate, butyl benzyl phthalate, diphenyl isophthalate, and dicyclohexyl phthalate (DPP, DBP, BBP, DPIP, and DCHP) from environmental water samples. The structure and morphology of the materials were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction(XRD). The parameters that influenced the enrichment performance such as amount of sorbent, sample pH, type of eluent, volume of eluent, salting-out effect, adsorption time, and desorption time were investigated. Under optimal conditions, excellent linear relationships were found in the concentration range from 0.5 to 100 μg L−1, the limits of detection (S/N = 3) were in the range of 0.26–0.45 μg L−1, and the intra-day and inter-day precisions (n = 6) were in the range of 3.7–4.8 and 3.2–4.3 %, respectively. The developed method was evaluated with real water samples, and satisfied spiked recoveries in the range of 99–104 % were achieved. The experimental results proved that Fe@SiO2@PEI had good adsorption for phthalate esters, and would be a good adsorbent for the magnetic solid-phase extraction of important pollutants from environmental water samples.ᅟ Graphical abstract A flowchart of the synthesis of polyethyleneimine grafted silica coated nanoscale zero valent iron (Fe@SiO2@PEI) and the process of the developed magnetic solid phase extraction of phthalate esters before the analysis by high performance liquid chromatography
Keywords: Fe@SiO2@PEI; Magnetic solid-phase extraction; Phthalate esters; High-performance liquid chromatography; Environmental water samples

The purpose of this work was to investigate the in vitro metabolism of nitracaine, a new psychoactive substance, using human liver microsome incubations, to evaluate the cytochrome P450 (CYP) enzyme isoforms responsible for the phase-I metabolism and to compare the information from the in vitro experiments with data resulting from an authentic user’s urine sample. Accurate mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and were used in the structural identification of metabolites. Two major and three minor phase-I metabolites were identified from the in vitro experiments. The observed phase-I metabolites were formed through N-deethylation, N,N-deethylation, N-hydroxylation, and de-esterification, with CYP2B6 and CYP2C19 being the main enzymes catalyzing their formation. One glucuronidated product was identified in the phase-II metabolism experiments. All of these metabolites are reported for the first time in this study except the N-deethylation product. All the in vitro metabolites except the minor N,N-deethylation product were also present in the human urine sample, thus demonstrating the reliability of the in vitro experiments in the prediction of the in vivo metabolism of nitracaine. In addition to the metabolites, three transformation products (p-nitrobenzoic acid, p-aminobenzoic acid, and 3-(diethylamino)-2,2-dimethylpropan-1-ol) were identified, as well as several glucuronides and glutamine derived of them.
Keywords: Nitracaine; High-resolution mass spectrometry; In vitro metabolism; Human liver microsomes; New psychoactive substance

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test by Natasha Yeow; Heather McLiesh; Liyun Guan; Wei Shen; Gil Garnier (5231-5238).
A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test
Keywords: Paper diagnostics; Indirect antiglobulin test; Coombs test; Blood typing; IgG; Antibody

Headspace-programmed temperature vaporization-mass spectrometry for the rapid determination of possible volatile biomarkers of lung cancer in urine by Ana Pérez Antón; Álvaro García Ramos; Miguel del Nogal Sánchez; José Luis Pérez Pavón; Bernardo Moreno Cordero; Ángel Pedro Crisolino Pozas (5239-5246).
We propose a new method for the rapid determination of five volatile compounds described in the literature as possible biomarkers of lung cancer in urine samples. The method is based on the coupling of a headspace sampler, a programmed temperature vaporizer in solvent-vent injection mode, and a mass spectrometer (HS-PTV-MS). This configuration is known as an electronic nose based on mass spectrometry. Once the method was developed, it was used for the analysis of urine samples from lung cancer patients and healthy individuals. Multivariate calibration models were employed to quantify the biomarker concentrations in the samples. The detection limits ranged between 0.16 and 21 μg/L. For the assignment of the samples to the patient group or the healthy individuals, the Wilcoxon signed-rank test was used, comparing the concentrations obtained with the median of a reference set of healthy individuals. To date, this is the first time that multivariate calibration and non-parametric methods have been combined to classify biological samples from profile signals obtained with an electronic nose. When significant differences in the concentration of one or more biomarkers were found with respect to the reference set, the sample is considered as a positive one and a new analysis was performed using a chromatographic method (HS-PTV-GC/MS) to confirm the result. The main advantage of the proposed HS-PTV-MS methodology is that no prior chromatographic separation and no sample manipulation are required, which allows an increase of the number of samples analyzed per hour and restricts the use of time-consuming techniques to only when necessary. Graphical abstract Schematic diagram of the developed methodology
Keywords: HS-PTV-MS; Volatile biomarkers; Multivariate calibration; Wilcoxon signed-rank test; Lung cancer and urine

A novel, highly selective, and sensitive resonance light scattering (RLS) detection approach coupled with high performance liquid chromatography (HPLC) was researched and developed for the synchronous analysis of three kinds of benzimidazole anthelmintics, including mebendazole (MBZ), albendazole (ABZ), and fenbendazole (FBZ) for the first time. In the pH range of 3.5–3.7 Britton-Robinson buffer medium, three kinds of anthelmintics, which were separated by HPLC, reacted with eosin Y (EY) to form 1:1 ion-association complexes, resulting in significantly enhanced RLS signals and the maximum peak located at 335 nm. The enhanced RLS intensity was in proportion to the MBZ, ABZ, and FBZ concentration in the range 0.2–25, 0.2–23, and 0.15–20 μg/mL, respectively. The limit of detection was in the range of 0.064–0.16 μg/mL. In addition, human urine was determined to validate the proposed method by spiked samples and real urine samples. Satisfactory results were obtained by HPLC-RLS method. Graphical Abstract The diagram mechanism of generating resonance between emitted light and scattered light
Keywords: Resonance light scattering; High performance liquid chromatography; Benzimidazole anthelmintics; Eosin Y

A method to detect small molecules with a molecularly imprinted polymer/quantum dot (MIP-QD) chip using a home-built optical fluidic system was first proposed in this study. Ractopamine (RAC) was used as the model molecule to demonstrate its feasibility. The sensing of the target molecule is based on the quenching amount of the quantum dots. The method is facile, cost-saving, easy for miniaturization and avoids the cumbersome steps that are needed to get the fluorescent quenching curve using a spectrofluorometer. Most importantly, more details and accurate response time can be obtained by use of this method. The experimental results show that the prepared chips with low cost are highly selective and the home-built detection system allows the fast binding kinetics. The recorded quenching process was used to study the kinetic uptake of RAC onto the MIP-QD chip and the specificity towards RAC. The system can further be utilized to study the effect of the solvent, pH and temperature on the selectivity of the prepared MIP. The methodology could be extended to other similar studies with different molecules. Graphical abstract Schematic illustration of the molecularly imprinted polymer/quantum dot chip capturing the target molecule
Keywords: Real-time fluorescence measurement; Molecular imprinting; Selective optosensing chip; Binding kinetics

SPR imaging biosensor for determination of laminin-5 as a potential cancer marker in biological material by A. Sankiewicz; L. Romanowicz; P. Laudanski; B. Zelazowska-Rutkowska; B. Puzan; B. Cylwik; E. Gorodkiewicz (5269-5276).
A new method for the selective determination of laminin-5 concentration using a biosensor and surface plasmon resonance imaging (SPRI) technique is presented. A biosensor based on the specific interaction of laminin-5 with rabbit polyclonal antibody was constructed. The analytically useful dynamic response range of the biosensor is between 0.014 and 0.1 ng mL−1. The detection limit is 4 pg mL−1. The potential influence of interferences on the SPRI signal was investigated, and the high selectivity of the biosensor was confirmed. In order to demonstrate the potential application of the biosensor, laminin-5 concentration in blood plasma was determined. The results were compared with the laminin-5 concentration obtained by the commercial enzyme-linked immunosorbent assay (ELISA) kit. A comparison of results from healthy donors obtained by SPRI measurement and ELISA indicates that they are close and shows good agreement with the data reported in the literature. The plasma samples of bladder cancer patients gave higher concentration measured with specific biosensor than by ELISA assay. The study shows the clear difference in concentration of laminin-5 in healthy humans and patients with bladder cancer. Extensive clinical studies using the newly developed method can result in an increase in the use of laminin-5 as a potential cancer marker.
Keywords: Laminin-5; Surface plasmon resonance imaging; Biosensors; Cancer marker

Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes by Raquel Vinhas; Cláudia Correia; Patricia Ribeiro; Alexandra Lourenço; Aida Botelho de Sousa; Alexandra R. Fernandes; Pedro V. Baptista (5277-5284).
Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome
Keywords: Chronic myeloid leukemia; Leukemia; RNA; Gold nanoparticles; Colorimetric detection

A new approach to design an efficient micropost array for enhanced direct-current insulator-based dielectrophoretic trapping by Mahdi Mohammadi; Mohammad Javad Zare; Hojjat Madadi; Jordi Sellarès; Jasmina Casals-Terré (5285-5294).
Direct-current insulator-based dielectrophoresis (DC-iDEP) is a well-known technique that benefits from the electric field gradients generated by an array of insulating posts to separate or trap biological particles. The aim of this study is to provide a first geometrical relationship of the post array that independent of the particles and/or medium, maximizes the trapping. A novel figure of merit is proposed to maximize the particle trapping in the post array while minimizing the required voltage, with a similar footprint and channel thickness. Different post array models with the variation of transversal distance (10 to 60 μm), longitudinal distance (10 to 80 μm), and post radius (10 to 150 μm) were analyzed using COMSOL Multiphysics finite element software. The obtained results indicated that a post radius of 40 μm larger than the transversal distance between posts could enhance the trapping condition between 56 % (for a transversal distance of 10 μm) and 341 % (for a transversal distance of 60 μm). For the validation of the numerical results, several microchannels with embedded post arrays were manufactured in polydimethylsiloxane (PDMS) and the particle trapping patterns of 6-μm-diameter polystyrene particles were measured experimentally. The experiments confirm the same trends as pointed out by the numerical analysis. The results show that this new figure of merit and geometrical relationship can be used to reduce the required electric field to achieve effective particle trapping and, therefore, avoid the negative effects of Joule heating in cells or viable particles. The main advantage of these results is that they depend only on the geometry of the micropost array and are valid for trapping different particles suspended in different media. Graphical abstract Analysis to maximize the particle trapping in the post array while minimizing the required voltage. I. Microfluidic channel design and experimental setup II. Numerical and experimental results. III. Maximum trapping value
Keywords: Dielectrophoresis; Modeling; Insulator-based dielectrophoresis; Particle trapping

The mechanism of cellular uptake by endocytosis and subsequent oxidative stress has been identified as the paradigm for the toxic response of cationically surface charged nanoparticles. In an attempt to circumvent the process, the effect of increased cellular membrane permeability on the uptake mechanisms of poly(amidoamine) dendrimers generations 4 (G4) and 6 (G6) in vitro was investigated. Immortalised, non-cancerous human keratinocyte (HaCaT) cells were treated with DL-buthionine-(S,R)-sulfoximine (BSO). Active uptake of the particles was monitored using fluorescence microscopy to identify and quantify endosomal activity and resultant oxidative stress, manifested as increased levels of reactive oxygen species, monitored using the carboxy-H2DCFDA dye. Dose-dependent cytotoxicity for G4 and G6 exposure was registered using the cytotoxicity assays Alamar Blue and MTT, from 6 to 72 h. Reduced uptake by endocytosis is observed for both dendrimer species. A dramatic change, compared to untreated cells, is observed in the cytotoxic and oxidative stress response of the BSO-treated cells. The significantly increased mitochondrial activity, dose-dependent antioxidant behaviour and reduced degree of endocytosis for both dendrimer generations, in BSO-treated cells, indicate enhanced permeability of the cell membrane, resulting in the passive, diffusive uptake of dendrimers, replacing endocytosis as the primary uptake mechanism. The complex MTT response reflects the importance of glutathione in maintaining redox balance within the mitochondria. The study highlights the importance of regulation of this redox balance for cell metabolism but also points to the potential of controlling the nanoparticle uptake mechanisms, and resultant cytotoxicity, with implications for nanomedicine.
Keywords: Dendrimer nanoparticles; PAMAM nanoparticles; Endocytosis; Passive diffusion; Oxidative stress; Membrane permeability

Threads for tear film collection and support in quantitative amino acid analysis by Vitaly Avilov; Qi Zeng; Scott A. Shippy (5309-5317).
The collection of tears for chemical composition analysis is complicated by both the difficulty in sampling the tear film and the relatively low microliter volumes available for analysis. The experiments in this study are focused on the demonstration of a method for determining amino acids from tear samples. Phenol red thread was used to absorptively collect tear fluid for qualitative and quantitative analyses of amino acids in basal, reflex, and emotional tears. The thread is also used as a support for sample preparation followed by elution with a buffer. The phenol red indicator on the thread turns from yellow to red with 15-s tear absorption and allows accurate volume measurement from 100 nL to over 1 μL. Derivatization of amino acids was performed directly on the thread with primary amine reactive fluorescamine for fluorescence detection. Analyte elution was performed via centrifugation with the thread in a pipet tip suspended in a centrifuge tube. Collected tear eluate was analyzed via capillary electrophoresis with LED-induced fluorescence. Glycine, glutamate, and aspartate were baseline resolved and used for method characterization. Recoveries were at 50 % for a single derivation and elution step but average recoveries near 90 % were found with two-step processing. Glutamate and aspartate are shown to be stable stored on thread for 3 days. Basal, reflex, and emotional tears were analyzed from three subjects showing distinct amino acid profiles for each tear type. The demonstration of this method may facilitate the development of routine tear compositional analysis to assess ocular health. Graphical Abstract Schematic drawing of thread-based tear collection and quantitative analysis
Keywords: Tear; Phenol red thread; Capillary electrophoresis; Reflex tears; Emotional tears; Amino acid

Hydrophilic molecularly imprinted polymers (H-MIP) with molecular recognition ability for iridoid glycosides (IGs) have been obtained via bulk polymerization combined with hydrolysis of ester groups. H-MIP were characterized by Fourier transform infrared spectroscopy (FT-IR). The hydrophilcity was measured by the contact angle measurement and the water dispersion stability. The obtained H-MIP demonstrated high selectivity and specific binding ability to five IGs in aqueous media. The group extraction efficiency of molecular imprinted solid-phase extraction (MISPE) for five IGs was investigated, including loading sample, breakthrough volume, washing solvent, and elution solvent. Compared with non-imprinted solid-phase extraction (NISPE), the higher average recovery (95.5 %) of five IGs with lower relative standard deviations values (below 6.1 %) using MISPE combined with high-performance liquid chromatography (HPLC) were achieved at three spiked levels in three blank samples. Under the optimum MISPE conditions, the wide linear range with the correlation coefficient of R 2  ≥ 0.9950 for five IGs with low limits of detection (LOD) and quantification (LOQ) (0.01–0.08 and 0.03–0.27 μg mL−1, respectively) were obtained. Chromatograms obtained using MISPE columns demonstrated that the matrix interference has been minimized and great interferences around IGs were also eliminated efficiently. These results indicated that the developed MISPE-HPLC method was selective, accurate, and applicable for the determination of IGs in water media. Graphical Abstract Preparation of hydrophilic molecularly imprinted polymers via bulk polymerization combined with hydrolysis of ester groups
Keywords: Molecularly imprinted polymers; HPLC; Natural products; Solid phase extraction; Iridoid glycosides

This study reports the development of a novel dummy template molecularly imprinted polymer (MIP)-coated barbell-shaped stir bar. The MIP stir bar coatings were prepared by using 2,2-bis(4-hydroxyphenyl)butane (BPB), 4,4'-dihydroxydiphenylmethane (BPF), 4-tert-butylphenol (PTBP), and tetrabromobisphenol A (TBBA) as dummy templates using a capillary in situ polymerization method. Uniform coatings can be prepared controllably. The method is simple, easy, and reproducible. The barbell-shaped stir bar was developed by using medical silicone tubes as wheels. The wheels could be removed and reinstalled when necessary; therefore, the barbell-shaped stir bar was easy to disassemble and reassemble. The novel MIP-coated stir bar showed good selectivity for the target analyte, bisphenol A (BPA). The established method is selective and sensitive with a lower detection limit for BPA of 0.003 μg/L. The dummy template MIP-coated stir bar is suitable for trace BPA analysis in real environmental water samples without template leakage. The novel stir bar can be used at least 100 times.
Keywords: Barbell-shaped stir bar; Stir bar sorptive extraction; Dummy template molecularly imprinted; Bisphenol A

Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors by Josep Ll. Acero Sánchez; Alex Fragoso; Hamdi Joda; Guillaume Suárez; Calum J. McNeil; Ciara K. O’Sullivan (5337-5346).
The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot
Keywords: SPR and electrochemical immunosensors; Site-directed protein modification; Surface chemistry; Self-assembled monolayer (SAM); Neuron-specific enolase (NSE)

A highly fluorescent nitrogen and sulfur codoped carbon nanoparticles (N,S-CNP) sample was obtained by microwave-assisted pyrolysis of citric acid and L-cysteine. After being purified by dialysis, the complexity and chemical composition of N,S-CNP were evaluated by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) as well as by UPLC coupled with ultraviolet (UV) absorption and fluorescence detection (UPLC-UV/FLD) methods. By using the high-resolution UPLC separation, the N,S-CNP were well fractionated into six fractions within 3.5 min. Based on high-accuracy MS and tandem (MS/MS) analyses, the N,S-CNP species were revealed to display various chemical formulas, including (C12H16N2O7S2) n , (C9H13NO8S) n , (C18H20N2O14S2) n , (C18H20N2O12S2) n , (C9H11NO5S) n , and (C9H11NO7S) n . More importantly, our study disclosed unambiguously for the first time that the N,S-CNP species exist as supramolecular clusters with their individual monomer units linked together through noncovalent bonding forces. By using UPLC-UV/FLD analysis, the spectral characteristics of each N,S-CNP species were revealed. Each individual CNP species possesses its unique absorption and PL properties with absorption bands that are redshifted, whereas its emission bands are blueshifted with its elution order. This work highlights the merit of UPLC-MS together with UPLC-UV/FLD to investigate the chemical composition and the spectral properties of each individual N,S-CNP species. It is anticipated that our proposed methodology will open up a new venue in optimizing experimental conditions for producing specific N,S-CNP species of desired composition. Graphical Abstract Carbon nanoparticles synthesized by microwave-assisted pyrolysis of citric acid and L-cysteine exist as supramolecular clusters with their individual monomer units linked together by noncovalent interactions
Keywords: Nitrogen and sulfur-codoped carbon nanoparticles; Noncovalent supramolecular clusters; Ultra-performance liquid chromatography; Mass spectrometry; Fluorescence

Identification of a ligand for tumor necrosis factor receptor from Chinese herbs by combination of surface plasmon resonance biosensor and UPLC-MS by Yan Cao; Ying-hua Li; Di-ya Lv; Xiao-fei Chen; Lang-dong Chen; Zhen-yu Zhu; Yi-feng Chai; Jun-ping Zhang (5359-5367).
Identification of bioactive compounds directly from complex herbal extracts is a key issue in the study of Chinese herbs. The present study describes the establishment and application of a sensitive, efficient, and convenient method based on surface plasmon resonance (SPR) biosensors for screening active ingredients targeting tumor necrosis factor receptor type 1 (TNF-R1) from Chinese herbs. Concentration-adjusted herbal extracts were subjected to SPR binding assay, and a remarkable response signal was observed in Rheum officinale extract. Then, the TNF-R1-bound ingredients were recovered, enriched, and analyzed by UPLC-QTOF/MS. As a result, physcion-8-O-β-d-monoglucoside (PMG) was identified as a bioactive compound, and the affinity constant of PMG to TNF-R1 was determined by SPR affinity analysis (K D  = 376 nM). Pharmacological assays revealed that PMG inhibited TNF-α-induced cytotoxicity and apoptosis in L929 cells via TNF-R1. Although PMG was a trace component in the chemical constituents of the R. officinale extract, it had considerable anti-inflammatory activities. It was found for the first time that PMG was a ligand for TNF receptor from herbal medicines. The proposed SPR-based screening method may prove to be an effective solution to analyzing bioactive components of Chinese herbs and other complex drug systems. Graphical abstract Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them. Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them
Keywords: Bioactive ingredients; Chinese herbs; UPLC-MS; Surface plasmon resonance; Tumor necrosis factor receptor

Cross-validation of a mass spectrometric-based method for the therapeutic drug monitoring of irinotecan: implementation of matrix-assisted laser desorption/ionization mass spectrometry in pharmacokinetic measurements by Eleonora Calandra; Bianca Posocco; Sara Crotti; Elena Marangon; Luciana Giodini; Donato Nitti; Giuseppe Toffoli; Pietro Traldi; Marco Agostini (5369-5377).
Irinotecan is a widely used antineoplastic drug, mostly employed for the treatment of colorectal cancer. This drug is a feasible candidate for therapeutic drug monitoring due to the presence of a wide inter-individual variability in the pharmacokinetic and pharmacodynamic parameters. In order to determine the drug concentration during the administration protocol, we developed a quantitative MALDI-MS method using CHCA as MALDI matrix. Here, we demonstrate that MALDI-TOF can be applied in a routine setting for therapeutic drug monitoring in humans offering quick and accurate results. To reach this aim, we cross validated, according to FDA and EMA guidelines, the MALDI-TOF method in comparison with a standard LC-MS/MS method, applying it for the quantification of 108 patients’ plasma samples from a clinical trial. Standard curves for irinotecan were linear (R 2 ≥ 0.9842) over the concentration ranges between 300 and 10,000 ng/mL and showed good back-calculated accuracy and precision. Intra- and inter-day precision and accuracy, determined on three quality control levels were always <12.8 % and between 90.1 and 106.9 %, respectively. The cross-validation procedure showed a good reproducibility between the two methods, the percentage differences within 20 % in more than 70 % of the total amount of clinical samples analysed.
Keywords: MALDI-TOF; Irinotecan; Bioanalytical methods; Drug monitoring/drug screening

Proteins are continuously exposed to various reactive chemical species (reactive oxygen/nitrogen species, endogenous/exogenous aldehydes/epoxides, etc.) due to physiological and chemical stresses, resulting in various chemical modifications such as oxidation, nitration, glycation/glycoxidation, lipidation/lipoxidation, and adduct formation with drugs/chemicals. Abundant proteins with a long half-life, such as hemoglobin (Hb, t 1/2 63 days, ∼150 mg/mL), are believed to be major targets of reactive chemical species that reflect biological events. Chemical modifications on Hb have been investigated mainly by mechanistic in vitro experiments or in vivo/clinical experiments focused on single target modifications. Here, we describe an optimized LC/ESI-SRM/MS method to screen oxidized, nitrated, lipidated, and glycated sites on Hb. In vivo preliminary results suggest that this method can detect simultaneously the presence of oxidation (+16 Da) of α-Met32, α-Met76, β-Met55, and β-Trp15 and adducts of malondialdehyde (+54 Da) and glycation (+162 Da) of β-Val1 in a blood sample from a healthy volunteer. Graphical Abstract Screening chemical modifications on hemoglobin
Keywords: Hemoglobin; Mass spectrometry; Chemical modification; Proteomics