Analytical and Bioanalytical Chemistry (v.409, #12)

A new biochromatography model based on DNA origami assembled PPARγ: construction and evaluation by Jie Zhou; Lingchang Meng; Chong Sun; Shanshan Chen; Fang Sun; Pei Luo; Yongxing Zhao (3059-3065).
As drug targets, receptors have potential to screen drugs. Silica is an attractive support to immobilize receptors; however, the lack of biocompatibility makes it easier for receptors to lose bioactivity, which remains an obstacle to its widespread use. With the advantage of biocompatibility, DNA origami can be used as a biological carrier to improve the biocompatibility of silica and assemble receptors. In this study, a new biochromatography model based on DNA origami was constructed. A large quantity of M13ssDNA was used as a scaffold, leading to significant costs, so M13ssDNA was self-produced from the bacteriophage particles. This approach is demonstrated using the ligand binding domain of gamma isoform peroxisome proliferator-activated receptor (PPARγ-LBD) as a research object. PPARγ-LBD was assembled on DNA origami carrier and then coupled on the surface of silica. The products were packed into the column as stationary phase to construct the biochromatography with the ability to recognize drugs. Affinity and specificity of the biochromatography model were evaluated by HPLC. The final results showed that the biochromatography could recognize rosiglitazone specifically, which further proved that the model could screen chemical compositions interacted with PPARγ. It was the first time to take advantage of DNA origami to assemble PPARγ to construct biochromatography. The new biochromatography model has the advantages of being efficient, convenient, and high-throughput. This method affords a new way to rapidly and conveniently screen active ingredients from complex sample plant extracts and natural product-like libraries.
Keywords: DNA origami; Biochromatography model; PPARγ; HPLC

is a graduate student in the laboratory of Nicholas Winograd at the Pennsylvania State University. Her research has focused on the use of secondary ion mass spectrometry for the imaging of biological systems and the visualization of exogenous compounds within single cells. is a research associate working in the Winograd group at the Pennsylvania State University. Her work focuses on high-resolution biological imaging using cluster secondary ion mass spectrometry and the development of gas cluster ion beams for the enhancement of ionization and reduction of matrix effects. is CEO of Nanopartz Inc., which was founded by him in 2006. The company has become the largest gold nanoparticle supplier to the research industry in the world, selling to more than 2000 customers in more than 30 countries. Its focus is to supply gold and other inorganic nanoparticles to the R & D community while spinning off venture-funded companies that will pursue specific applications in nanotechnology. is the Evan Pugh University Professor of Chemistry at the Pennsylvania State University. His group focuses on the use of secondary ion mass spectrometry in imaging biomaterials and single cells and studying fundamental aspects of ion/solid interactions and the characterization of solid surfaces. Obtaining a comprehensive grasp of the behavior and interaction of pharmaceutical compounds within single cells provides some of the fundamental details necessary for more effective drug development. In particular, the changes ensuing in the carrier, drug, and host environment in targeted drug therapy applications must be explored in greater detail, as these are still not well understood. Here, nilotinib-functionalized gold nanoparticles are examined within single mammalian cells with use of imaging cluster secondary ion mass spectrometry in a model study designed to enhance our understanding of what occurs to these particles once that have been internalized. Nilotinib, several types of gold nanoparticles, and the functionalized combination of the two were surveyed and successfully imaged within single cells to determine uptake and performance. Both nilotinib and the gold particle are able to be distinguished and visualized in the functionalized nanoparticle assembly within the cell. These compounds, while both internalized, do not appear to be present in the same pixels of the chemical image, indicating possible cleavage of nilotinib from the particle after cell uptake. The method provided in this work is a direct measurement of uptake and subcellular distribution of an active drug and its carrier within a framework. The results obtained from this study have the potential to be applied to future studies to provide more effective and specific cellular delivery of a relevant pharmaceutical compound.
Keywords: Secondary ion mass spectrometry; Targeted drug therapy; Nilotinib; Gold nanoparticles

Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides by Zixiang Yu; Xinyuan Zhao; Fang Tian; Yang Zhao; Yong Zhang; Yi Huang; Xiaohong Qian; Wantao Ying (3077-3087).
Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in 18O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC–MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples. Graphical abstract Through integrating the information of intact glycopeptide, fragment ions filters and endoglycosidase digestion, the reliability of the identification could be significantly improved. We quantified the site-specific glycoforms occupancy ratios through the MS response signaling of each glycopeptide at the same time.
Keywords: Glycoprotein; Glycosylation; HILIC; Mass spectrometry; Byonic

In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry by Zoltan Szabo; James R. Thayer; Yury Agroskin; Shanhua Lin; Yan Liu; Kannan Srinivasan; Julian Saba; Rosa Viner; Andreas Huhmer; Jeff Rohrer; Dietmar Reusch; Rania Harfouche; Shaheer H. Khan; Christopher Pohl (3089-3101).
Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS2 in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS2 permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.
Keywords: High performance anion exchange chromatography-pulsed amperometric detection (HPAE-PAD); Native N-linked glycans; Glycan annotation; Mass spectrometry; Hyphenation

Magnetic melamine-formaldehyde resin was prepared via water-in-oil emulsification approach by entrapping Fe3O4 magnetic nanoparticles as the core. The preparation of the magnetic resin was optimized by investigating the amount of polyethylene glycol 20000 and Fe3O4 nanoparticles, the concentration of the catalyst (hydrochloric acid), as well as the mechanical stirring rate. The prepared material was characteristic of excellent anion-exchange capacity, good water wettability, and proper magnetism. Its application was demonstrated by magnetic solid-phase extraction of nonsteroidal anti-inflammatory drugs coupled to high performance liquid chromatography-UV analysis. Under the optimal conditions, the proposed method showed broad linear range of 1–5000 ng mL–1 of milk and urine samples, satisfactory reproducibility with intra-day and inter-day relative standard deviations less than 12.4% and 9.7%, respectively, and low limits of detection of 0.2 ng mL–1 for the studied nonsteroidal anti-inflammatory drugs. The developed method was successfully used for the determination of the nonsteroidal anti-inflammatory drugs in spiked urine and milk samples. The magnetic melamine-formaldehyde resin was promising for the sample pretreatment of acidic analytes via anion-exchange interaction with convenient operation from complex sample matrix. Graphical abstract Magnetic solid-phase extraction based on melamine-formaldehyde resin
Keywords: Melamine-formaldehyde resin; Magnetic solid-phase extraction; Nonsteroidal anti-inflammatory drugs; Sample pretreatment

Tackling saponin diversity in marine animals by mass spectrometry: data acquisition and integration by Corentin Decroo; Emmanuel Colson; Marie Demeyer; Vincent Lemaur; Guillaume Caulier; Igor Eeckhaut; Jérôme Cornil; Patrick Flammang; Pascal Gerbaux (3115-3126).
Saponin analysis by mass spectrometry methods is nowadays progressively supplementing other analytical methods such as nuclear magnetic resonance (NMR). Indeed, saponin extracts from plant or marine animals are often constituted by a complex mixture of (slightly) different saponin molecules that requires extensive purification and separation steps to meet the requirement for NMR spectroscopy measurements. Based on its intrinsic features, mass spectrometry represents an inescapable tool to access the structures of saponins within extracts by using LC-MS, MALDI-MS, and tandem mass spectrometry experiments. The combination of different MS methods nowadays allows for a nice description of saponin structures, without extensive purification. However, the structural characterization process is based on low kinetic energy CID which cannot afford a total structure elucidation as far as stereochemistry is concerned. Moreover, the structural difference between saponins in a same extract is often so small that coelution upon LC-MS analysis is unavoidable, rendering the isomeric distinction and characterization by CID challenging or impossible. In the present paper, we introduce ion mobility in combination with liquid chromatography to better tackle the structural complexity of saponin congeners. When analyzing saponin extracts with MS-based methods, handling the data remains problematic for the comprehensive report of the results, but also for their efficient comparison. We here introduce an original schematic representation using sector diagrams that are constructed from mass spectrometry data. We strongly believe that the proposed data integration could be useful for data interpretation since it allows for a direct and fast comparison, both in terms of composition and relative proportion of the saponin contents in different extracts. Graphical Abstract A combination of state-of-the-art mass spectrometry methods, including ion mobility spectroscopy, is developed to afford a complete description of the saponin molecules in natural extracts
Keywords: Triterpene glycosides; Echinoderms; Sea cucumbers; Mass spectrometry; Natural products; Ion mobility; MALDI-ToF; LC-MS

Solid-phase microextraction with polysulfone and molecularly imprinted polymers as coating on nickel foam were used to adsorb and enrich floxacin drugs. The preparation method is simple and reproducible to obtain the materials with controlled thickness. After evaluation by scanning electron microscope and various adsorption experiments, the materials were used to adsorb analytes in water samples and biological samples. Coupling with chromatographic analysis, the method recoveries are satisfactory with 90.0–104.8% and 79.31–107.1% for water and biological samples. The method repeatability by intra- and interday experiments shows that the RSD values for water and biological samples were 1.0–9.9% and 1.7–10.3%, with the quantitative limits of three floxacin drugs as 3.0–6.2 μg L−1. Graphical Abstract Preparation diagram of polysulfone material
Keywords: Polysulfone; Molecularly imprinted polymers; Solid-phase microextraction; Floxacin drugs

Hydrazide-functionalized affinity on conventional support materials for glycopeptide enrichment by Muhammad Salman Sajid; Fahmida Jabeen; Dilshad Hussain; Muhammad Naeem Ashiq; Muhammad Najam-ul-Haq (3135-3143).
In affinity chromatography, enrichment of biomolecules is dependent on the selection of affinity sites immobilized onto a suitable support material. A few hydrazide - functionalized materials with surface modification protocols compatible to conventional support materials like silica and cellulose are reported. The study demonstrates the modification/derivatization pathways that can be adopted to modify the support materials with similar surface chemistry like cellulose, poly(GMA/DVB), or diamond. Poly(GMA/DVB) and cellulose represent hydrophilic supports whereas diamond is a hydrophobic support material. SEM images of three materials provide surface morphology whereas FT-IR confirms reaction completion and derivatization. These hydrazide - functionalized materials are applied to fetuin digest for glycopeptides enrichment and subsequently for selectivity and sensitivity assessment. Statistically, poly(GMA/DVB) shows 85.7% sensitivity with specificity of 88.8% in the enrichment experiments. Diamond offers hydrophobic interactions to non-glycopeptides and they co-elute with glycopeptides, resulting in reduced sensitivity down to 69.2%. Poly(GMA/DVB) shows recovery up to 89%, while recovery for cellulose and diamond is 83 and 71%, respectively. The materials enrich mono-N-linked-glycosylated peptide from tryptic digest of chicken avidin spiked in fetuin digest. The hydrazide group density on cellulose, poly(GMA/DVB), and diamond is 2.8, 2.3, and 2.1 mmol/g, respectively; this contributes towards the specificity and sensitivity of designed materials. The materials are also applied to serum samples and enriched glycopeptides characteristic of serum glycoproteins of clinical importance. Therefore this study provides routes for the economical surface modifications of support materials and to fabricate affinity materials with improved efficiency. Graphical Abstract Glycopeptides enrichment by hydrazine affinity
Keywords: Glycopeptides; Hydrazine; Cellulose; Diamond; Poly(GMA/DVB); MALDI-MS

Signal Enhancement in the HPLC-ESI-MS/MS analysis of spironolactone and its metabolites using HFIP and NH4F as eluent additives by Kalev Takkis; Rudolf Aro; Lenne-Triin Kõrgvee; Heili Varendi; Jana Lass; Koit Herodes; Karin Kipper (3145-3151).
This paper describes an LC-MS/MS method to determine the concentration of spironolactone and its metabolites 7-alpha-methylthiospironolactone and canrenone in blood plasma samples. The resulting assay is simple (using protein precipitation for sample preparation) and sensitive (the lower limit of quantification is close to 0.5 ng/ml) while requiring only 50 μl of plasma, making it especially suitable for analyzing samples obtained from pediatric and neonatal patients where sample sizes are limited. The sensitivity is achieved by using ammonium fluoride as an eluent additive, which in our case amplifies the signal from our analytes in the plasma solution on average about 70 times. The method is fully validated according to the European Medicines Agency’s guideline and used for the measurement of pediatric patients’ samples in clinical trials for evaluating oral spironolactone’s and its metabolites’ pharmacokinetics in children up to 2 years of age.
Keywords: Spironolactone; Ammonium fluoride; Signal enhancement; Liquid chromatography-mass spectrometry; Ionization efficiency; Hexafluoroisopropanol

Leaner and greener analysis of cannabinoids by Elizabeth M. Mudge; Susan J. Murch; Paula N. Brown (3153-3163).
There is an explosion in the number of labs analyzing cannabinoids in marijuana (Cannabis sativa L., Cannabaceae) but existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging solvents. The objective of this work was to develop and validate an accurate method for analyzing cannabinoids in cannabis raw materials and finished products that is more efficient and uses fewer toxic solvents. An HPLC-DAD method was developed for eight cannabinoids in cannabis flowers and oils using a statistically guided optimization plan based on the principles of green chemistry. A single-laboratory validation determined the linearity, selectivity, accuracy, repeatability, intermediate precision, limit of detection, and limit of quantitation of the method. Amounts of individual cannabinoids above the limit of quantitation in the flowers ranged from 0.02 to 14.9% w/w, with repeatability ranging from 0.78 to 10.08% relative standard deviation. The intermediate precision determined using HorRat ratios ranged from 0.3 to 2.0. The LOQs for individual cannabinoids in flowers ranged from 0.02 to 0.17% w/w. This is a significant improvement over previous methods and is suitable for a wide range of applications including regulatory compliance, clinical studies, direct patient medical services, and commercial suppliers.
Keywords: Green chemistry; Single-laboratory validation; Cannabis ; Cannabinoids; Medical marijuana

A novel two-step sequential extraction has been developed to assess the bioaccessibility of As, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in airborne particulate matter following inhalation and transport into the human gastrointestinal tract by mucociliary clearance. A new artificial mucus fluid (AMF) was used to determine the bioaccessible potentially toxic element (PTE) fraction in the upper airways, in sequence with the simplified bioaccessibility extraction test (SBET) or the stomach phase of the unified bioaccessibility method (gastric fluid only) (UBMG). Filter dynamic measurement system TX40 filters smeared with soil reference material (BGS RM 102) were used as test samples. Analysis was performed by ICP-MS. Comparison between results obtained for soil alone and when the soil was supported on TX40 filters indicated that the presence of the substrate did not affect the extraction efficiency, although a large Zn blank was detected. The sequential AMF→SBET extraction liberated similar amounts of Fe, Mn, Ni and Zn to the SBET alone; but significantly less Cd; and significantly more As, Cr, Cu and Pb. The sequential AMF→UBMG extraction liberated similar amounts of Cd, Cr, Mn and Zn to the UBMG alone, but significantly less As, Fe and Ni; and significantly more Cu and Pb. Enhanced extractability was due to the greater quantities of exchangeable ions and complexing agents present. Adoption of a two-step sequential extraction (AMF followed by either the SBET or the UBMG) is recommended because it is more representative of biological conditions and avoids overestimation or underestimation of bioaccessible PTE concentrations. Graphical Abstract Simulated PM10 sample: BGS RM 102 ironstone soil on TX40 filter
Keywords: Bioaccessibility; Inhaled particulate matter; Potentially toxic elements; Artificial mucus fluid

Isatin functionalized nanoporous SBA-15 as a selective fluorescent probe for the detection of Hg(II) in water by Negar Lashgari; Alireza Badiei; Ghodsi Mohammadi Ziarani; Farnoush Faridbod (3175-3185).
A highly ordered mesoporous silica material functionalized with isatin (SBA-Pr-IS) was designed and synthesized. Characterization techniques including XRD, TGA, BET, SEM, and FT-IR were employed to characterize the pore structure, textural properties, microscopic morphology, and molecular composition of grafted organic moieties of SBA-Pr-IS. The successful attachment of the organic moiety (0.34 mmol g−1) without the SBA-15 structure collapsing after the modification steps was confirmed. Fluorescence characterization of SBA-Pr-IS was examined upon addition of a wide variety of cations in aqueous medium and it showed high sensitivity toward Hg2+ ions. During testing in an ion competition experiment, it was observed that the fluorescence changes of the probe were remarkably specific for Hg2+ ions. Furthermore, a good linearity between the fluorescence intensity of this material and the concentration of Hg2+ ions was constructed with a suitable detection limit of 3.7 × 10−6 M. Finally, the applicability of the proposed method was successfully evaluated for the determination of Hg2+ ions in real samples. Therefore, SBA-Pr-IS can be used as an efficient fluorescence probe for Hg2+ ions. Graphical Abstract A novel organic-inorganic hybrid material was designed and synthesized by functionalization of SBA-15 mesoporous silica material with isatin. The evaluation of the sensing ability of SBA-Pr-IS using fluorescence spectroscopy revealed that the SBA-Pr-IS was a selective fluorescent probe for Hg2+ ion in water in the presence of a wide range of metal cations.
Keywords: SBA-15; Fluorescent probe; Nanoporous silica; Hg2+ ion; Isatin

A new analytical approach, based on micro-transflection measurements from a diamond-coated metal sampling stick, is presented for the analysis of painting varnishes. Minimally invasive sampling is performed from the varnished surface using the stick, which is directly used as a transflection substrate for micro Fourier transform infrared (FTIR) measurements. With use of a series of varnished model paints, the micro-transflection method has been proved to be a valuable tool for the identification of surface components thanks to the selectivity of the sampling, the enhancement of the absorbance signal, and the easier spectral interpretation because the profiles are similar to transmission mode ones. Driven by these positive outcomes, the method was then tested as tool supporting noninvasive reflection FTIR spectroscopy during the assessment of varnish removal by solvent cleaning on paint models. Finally, the integrated analytical approach based on the two reflection methods was successfully applied for the monitoring of the cleaning of the sixteenth century painting Presentation in the Temple by Vittore Carpaccio. Graphical Abstract Micro-transflection FTIR on a metallic stick for the identification of varnishes during painting cleanings
Keywords: Varnishes; Micro fourier transform infrared spectroscopy; Reflection spectroscopy; Artworks; Cleaning

New sampling device for on-site measurement of SVOC gas-phase concentration at the emitting material surface by Mylène Ghislain; Joana Beigbeder; Hervé Plaisance; Valérie Desauziers (3199-3210).
The gas-phase concentration at the material surface (y 0 ) is pointed out in the literature as a key parameter to describe semivolatile organic compound (SVOC) emissions from materials. This is an important input data in predictive models of SVOC behavior indoors and risk exposure assessment. However, most of the existing measurement methods consist of determining emission rates and not y 0 and none allow on-site sampling. Hence, a new passive sampler was developed. It consists of a glass cell that is simply placed on the material surface until reaching equilibrium between material and air; y 0 is then determined by solid-phase microextraction (SPME) sampling and GC-MS analysis. The limits of detection are at the μg/m3 level and relative standard deviations (RSD) below 10%. A variation of 11% between two sets of experiments involving different cell volumes confirmed the y 0 measurement. In addition, due to the ability of SVOCs to be sorbed on surfaces, the cell wall/air partition was assessed by determining the inner cell surface concentration of SVOCs, which is the concentration of SVOCs adsorbed on the glass, and the cell surface/air partition coefficient (K glass ). The recovery yields of the SVOCs sorbed on the cell walls are strongly compound-dependent and comprise between 2 and 93%. The K glass coefficients are found to be lower than the stainless steel/air partition coefficient (K ss ), showing that glass is suitable for the SVOC sampling. This innovative tool opens up promising perspectives in terms of identification of SVOC sources and quantification of their emissions indoors, and would significantly contribute to human exposure assessment. Graphical Abstract Passive sampling for the determination of SVOCs concentration at the material/air interface
Keywords: Material/air interface concentration (y 0 ); SVOCs; Emission cell; Indoor air; Organophosphate esters; Flame retardants

Lipidomic analysis of plasma in patients with lacunar infarction using normal-phase/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry by Li Yang; Pu Lv; Wanpeng Ai; Linnan Li; Sensen Shen; Honggang Nie; Yabing Shan; Yu Bai; Yining Huang; Huwei Liu (3211-3222).
Stroke is a major cause of mortality and long-term disability worldwide. The study of biomarkers and pathogenesis is vital for early diagnosis and treatment of stroke. In the present study, a continuous-flow normal-phase/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (NP/RP 2D LC-QToF/MS) method was employed to measure lipid species in human plasma, including healthy controls and lacunar infarction (LI) patients. As a result, 13 lipid species were demonstrated with significant difference between the two groups, and a “plasma biomarker model” including glucosylceramide (38:2), phosphatidylethanolamine (35:2), free fatty acid (16:1), and triacylglycerol (56:5) was finally established. This model was evaluated as an effective tool in that area under the receiver operating characteristic curve reached 1.000 in the discovery set and 0.947 in the validation set for diagnosing LI patients from healthy controls. Besides, the sensitivity and specificity of disease diagnosis in validation set were 93.3% and 96.6% at the best cutoff value, respectively. This study demonstrates the promising potential of NP/RP 2D LC-QToF/MS-based lipidomics approach in finding bio-markers for disease diagnosis and providing special insights into the metabolism of stroke induced by small vessel disease. Graphical abstract Flow-chart of the plasma biomarker model establishment through biomarker screening and validation
Keywords: Stroke; Lacunar infarction; Lipidomics; Biomarker; NP/RP 2D LC-QToF/MS

Nitrogen oxides as dopants for the detection of aromatic compounds with ion mobility spectrometry by Urszula Gaik; Mika Sillanpää; Zygfryd Witkiewicz; Jarosław Puton (3223-3231).
Limits of detection (LODs) in ion mobility spectrometry (IMS) strictly depend on ionization of the analyte. Especially challenging is ionization of compounds with relatively low proton affinity (PA) such as aromatic compounds. To change the course of ion-molecule reactions and enhance the performance of the IMS spectrometer, substances called dopants are introduced into the carrier gas. In this work, we present the results of studies of detection using nitrogen oxides (NOx) dopants. Three aromatic compounds, benzene, toluene, toluene diisocyanate and, for comparison, two compounds with high PA, dimethyl methylphosphonate (DMMP) and triethyl phosphate (TEP), were selected as analytes. The influence of water vapour on these analyses was also studied. Experiments were carried out with a generator of gas mixtures that allowed for the simultaneous introduction of three substances into the carrier gas. The experiments showed that the use of NOx dopants significantly decreases LODs for aromatic compounds and does not affect the detection of compounds with high PA. The water vapour significantly disturbs the detection of aromatic compounds; however, doping with NOx allows to reduce the effect of humidity. Graphical Abstract Two possible ionization mechanisms of aromatic compounds in ion mobility spectrometry: proton transfer reaction and adduct formation
Keywords: Ion mobility spectrometry; Dopants; Aromatic compounds; Nitrogen oxides

In this paper, the effect of isotope-labeled analogs on the liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) measurement was evaluated based on the comparison research of electrospray ionization responses (ESI) and matrix effect of melamine, 13C3-melamine, 13C3+15N3-melamine, and 15N3-melamine. The isotope-labeled melamines had similar ionization efficiency with melamine in the electrospray ionization source, but the intensity of corresponding quantitative fragment ions had distinctive differences. Based on the density functional theory at the B3LYP/6-311+G** level, this phenomenon was explained very well. The rare cleavage pathways of melamine, which just could be exactly identified by 15N-labeled melamines, resulted in the difference of quantitative fragment ions between 15N-labeled melamines and melamine. The interaction of ESI response between melamine and isotope-labeled melamines was investigated using MRM monitor mode. 15N-labeled melamine had significant ion inter-suppression effect on melamine, while 13C-labeled melamine had little influence on melamine. Finally, the influence of different isotope-labeled melamines on the LC-IDMS result was evaluated using the IDMS correction factor (θ). Taking the determination of melamine in milk powder as an example, the matrix effects of different isotope-labeled melamines and melamine had notable difference and the impact of this difference on the measurement results depended on the concentrations of analyte and matrix solution. It was worth noting that 15N3-melamine exhibited significant ion suppression to melamine in matrix solution. The deviation of the results from IDMS method might reach 59% using 15N3-melamine as internal standard in special matrix solution. Graphical Abstract The comparison of ESI responses of melamine, 13C3-melamine, 13C3+15N3-melamine and 15N3-melamine
Keywords: Comparison; ESI responses; Matrix effect; Melamine; Isotope-labeled melamines; LC-IDMS

An ultrasensitive electrochemical immunosensor for the detection of prostate-specific antigen based on conductivity nanocomposite with halloysite nanotubes by Yueyuan Li; Malik Saddam Khan; Lihui Tian; Li Liu; Lihua Hu; Dawei Fan; Wei Cao; Qin Wei (3245-3251).
A sensitive label-free amperometric electrochemical immunosensor for detection of prostate-specific antigen (PSA) was proposed in this work. The nanocomposite of halloysite nanotubes with polypyrrole shell and palladium nanoparticles (HNTs@PPy-Pd) was used as a novel signal label. The HNTs with adequate hydroxyl groups are economically available raw materials. PPy, as an electrically conducting polymer material, can be absorbed to the surface of HNTs by in situ oxidative polymerization of the pyrrole monomer and form a shell on the HNTs. The shell of PPy could not only improve the conductivity of the nanocomposite but also absorb large amounts of Pd nanoparticles (NPs). The Pd NPs with high electrocatalytic activity toward the reduction of H2O2 and the HNTs@PPy-Pd nanocomposite as the analytical signal label could improve the sensitivity of the immunosensor. Under optimal conditions, the immunosensor showed a low detection limit (0.03 pg/mL) and a wide linear range (0.0001 to 25 ng/mL) of PSA. Moreover, its merits such as good selectivity, acceptable reproducibility, and stability indicate that the fabricated immunosensor has a promising application potential in clinical diagnosis. Graphical Abstract A new label-free amperometric electrochemical immunosensor based on HNTs@PPy-Pd nanocomposite for quantitative detection of PSA.
Keywords: Halloysite nanotubes; Prostate-specific antigen; Polypyrrole shell; Label-free immunosensor

We report on application of conductive silver paste smeared glass slides as Raman spectroscopy sample substrates for label-free detection of HIV-1 p24 antigen in blood plasma. We also show that the same substrates can be applied in Raman spectroscopic screening of blood plasma for presence of HIV. The characteristic Raman spectrum of HIV-1 p24 antigen displayed prominent bands that were assigned to ribonucleic acids (RNA) and proteins that constitute the antigen. This spectrum can be used as reference during Raman spectroscopic screening for HIV in plasma within the first few days after exposure (<7 days). The Raman spectra obtained from HIV+ plasma displayed unique peaks centered at wavenumbers 928, 990, 1270, 1397, and 1446 cm−1 attributed to the Raman active vibrations in the virion carbohydrates, lipids, and proteins. Other bands similar to those reported in literature were also seen and assignments made. The attachment of the HIV virions to silver nanoparticles via gp120 glycoprotein knobs was thought to be responsible for the enhanced Raman signals of proteins associated with the virus. The principal component analysis (PCA) applied on the combined spectral data showed that HIV− and HIV+ spectra had differing spectral patterns. This indicated the great power of Raman spectroscopy in HIV detection when plasma samples are deposited onto silver paste smeared glass substrates. The Raman peaks responsible for the segregation of the spectral data in PCA were mainly those assigned to the viral proteins (645, 725, 813, 1270, and 1658 cm−1). Excellent results were obtained from Artificial Neural Network (ANN) applied on the HIV+ Raman spectral data around the prominent peak centered at 1270 cm−1 with R (coefficient of correlation) and R 2 (coefficient of determination) values of 0.9958 and 0.9895, respectively. The method has the potential of being used as quick blood screening for HIV before blood transfusion with the Raman peaks assigned to the virion proteins acting as reference. Graphical Abstract The HIV type 1 virus particle gets attached to the silver nanoparticle contained in the conductive silver paste smear onto a glass slide. This results in strong Raman signals associated with the components of the virion. The signals are collected, dispersed in a spectrometer and displayed on a computer screen. Method can be used as a label-free and rapid HIV screening in blood plasma
Keywords: Raman spectroscopy; HIV-1 p24 antigen; HIV positive plasma; Principal component analysis; Artificial Neural Networks

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection by Jonathan Sabaté del Río; Ivan Magriñà Lobato; Olena Mayboroda; Ioanis Katakis; Ciara K. O’Sullivan (3261-3269).
Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10−15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.
Keywords: Electrochemical genosensor; Solid-phase recombinase polymerase amplification; Surface chemistry; Real samples from hares

High throughput and automatic colony formation assay based on impedance measurement technique by Kin Fong Lei; Chich-Hao Kao; Ngan-Ming Tsang (3271-3277).
To predict the response of in vivo tumors, in vitro culture of cell colonies was suggested to be a standard assay to achieve high clinical relevance. To describe the responses of cell colonies, the most widely used quantification method is to count the number and size of cell colonies under microscope. That makes the colony formation assay infeasible to be high throughput and automated. In this work, in situ analysis of cell colonies suspended in soft hydrogel was developed based on impedance measurement technique. Cell colonies cultured between a pair of parallel plate electrodes were successfully analyzed by coating a layer of base hydrogel on one side of electrode. Real-time and label-free monitoring of cell colonies was realized during the culture course. Impedance magnitude and phase angle respectively represented the summation effect of colony responses and size of colonies. In addition, dynamic response of drug-treated colonies was demonstrated. High throughput and automatic colony formation assay was realized to facilitate more objective assessments in cancer research. Graphical Abstract High throughput and automatic colony formation assay was realized by in situ impedimetric analysis across a pair of parallel plate electrodes in a culture chamber. Cell colonies suspended in soft hydrogel were cultured under the tested substance and their dynamic response was represented by impedance data.
Keywords: Impedance measurement; Parallel plate electrodes; Colony formation assay; 3D cell culture; Chemosensitivity