Aptamer based hybrid monolithic pipette tips supported by melamine sponge for enrichment of proteins.

BACKGROUND: The convenient preparation and application of functionalized organic-inorganic hybrid monolithic materials have obtained substantial interest in the pretreatment of complex samples by solid-phase extraction (SPE). Compared to the in-tube solid-phase microextraction in fused-silica capillaries, micro SPE in plastic pipette tips have fascinating merits for the easily operated enrichment of trace target analytes from biological samples. However, the poor compatibility of organic-inorganic hybrid monoliths with plastics leads to the rare appearance of commercial hybrid monolithic pipette tips (HMPTs). Therefore, how to synthesize the organic-inorganic hybrid monolithic materials with better extraction performance in plastic pipette tips becomes a challenge. RESULTS: We develop a facile and cheap strategy to immobilize organic-inorganic hybrid monoliths in pipette tips. Melamine sponge was employed as the supporting skeleton to in situ assemble amine- and thiol-bifunctionalized hybrid monolithic material via "one pot" in a pipette tip, and gold nanoparticles (GNPs) and thiol-modified aptamer against human α-thrombin were sequentially attached to the hybrid monolith within the HMPTs. The average coverage density of the aptamer with GNPs as an intermediary reached as high as 818.5 pmol µL-1. The enriched thrombin concentration was determined by a sensitive enzymatic chromogenic assay with the limit of detection of 2 nM. The extraction recovery of thrombin at 10 nM in human serum was 86.1 % with a relative standard deviation of 6.1 %. This proposed protocol has been applied to the enrichment and determination of thrombin in real serum sample with strong anti-interference ability, low limit of detection and high recovery. SIGNIFICANCE: The amine- and thiol-bifunctionalized HMPTs prepared with sponge as the skeleton frame provided a novel substrate material to decorate aptamers for efficient enrichment of proteins. This enlightens us that we can take advantage of the tunability of sponge assisted HMPTs to produce and tailor a variety of micro SPE pipette tips for broader applications on the analysis of trace targets in complex biological, clinic and environmental samples.

A novel zwitterionic magnetic nanocomposite developed for non-invasive speciation analysis of inorganic chromium.

3-(2-Aminoethylamino)propyltriethoxysilane and carboxyethylsilanetriol sodium salt were grafted on silica-coated Fe3O4 nanoparticles via sol-gel process to prepare novel amine- and carboxyl-bifunctionalized magnetic nanocomposites (SMNPs-(NH2 + COOH)). After well characterized, this doubly functionalized material was used as magnetic solid-phase extraction (MSPE) adsorbent to separate and enrich inorganic chromium species followed by inductively coupled plasma-mass spectrometry detection. The optimization of MSPE operation parameters including pH was conducted. It is reasonably elucidated that the adsorption mechanisms of zwitterionic SMNPs-(NH2 + COOH) towards chromium species are electrostatic and/or coordination interactions. Cr(VI) and Cr(III) can be adsorbed around pH 3.0 and around 10.0 respectively with strong anti-interference ability not only from other co-existing ions but also from the two labile species each other, and eluted by dilute nitric acid solution. With a 15-fold enrichment factor, the limits of detection of Cr(VI) and Cr(III) were 0.008 and 0.009 µg L-1, respectively, profiting from the maximum adsorption capacities of 7.52 and 6.11 mg g-1. The just one magnetic extraction matrix based speciation scheme possesses excellent convenience and friendliness to Cr(VI) and Cr(III) without any oxidation or reduction prior to capture of these two species. This protocol has been successfully applied to the speciation analysis of inorganic chromium in real-world environmental water samples.

Effect of ionic liquids as mobile phase additives on retention behaviors of G-quadruplexes in reversed-phase high performance liquid chromatography.

G-quadruplexes (G4s) play an important role in a variety of biological processes and have extensive application prospects. Due to the significance of G4s in physiology and biosensing, studies on G4s have attracted much attention, stimulating the development or improvement of methods for G4 structures and polymorphism analysis. In this work, ionic liquids (ILs) were involved as mobile phase additives in reversed-phase high performance liquid chromatography (RP-HPLC) to analyse G4s with various conformations for the first time. How ILs affected the retention behaviors of G4s was investigated comprehensively. It was found that the addition of ILs markedly enhanced G4 retention, along with obvious amelioration on chromatographic peak shapes and separation. The influence of pH of mobile phase and types of ILs were also included in order to acquire an in-depth understanding. It appeared that the effect of ILs on G4 retention behaviors was the result of a combination of various interactions between G4s with the hydrophobic stationary phase and with the IL-containing mobile phase, where ion pair mechanism and enhanced hydrophobic interaction dominated. The findings of this work revealed that ILs could effectively improve the separation of G4s in RP-HPLC, which was conducive to G4 structural analysis, especially for G4s polymorphism elucidation.

Prediction of the n-Octanol/Water Partition Coefficients of Basic Compounds Using Multi-Parameter QSRR Models Based on IS-RPLC Retention Behavior in a Wide pH Range.

The n-octanol-water partition coefficient (logP) is an important physicochemical parameter which describes the behavior of organic compounds. In this work, the apparent n-octanol/water partition coefficients (logD) of basic compounds were determined using ion-suppression reversed-phase liquid chromatography (IS-RPLC) on a silica-based C18 column. The quantitative structure-retention relationship (QSRR) models between logD and logkw (logarithm of retention factor corresponding to 100% aqueous fraction of mobile phase) were established at pH 7.0-10.0. It was found that logD had a poor linear correlation with logkw at pH 7.0 and pH 8.0 when strongly ionized compounds were included in the model compounds. However, the linearity of the QSRR model was significantly improved, especially at pH 7.0, when molecular structure parameters such as electrostatic charge ne and hydrogen bonding parameters A and B were introduced. External validation experiments further confirmed that the multi-parameter models could accurately predict the logD value of basic compounds not only under strong alkaline conditions, but also under weak alkaline and even neutral conditions. The logD values of basic sample compounds were predicted based on the multi-parameter QSRR models. Compared with previous work, the findings of this study extended the pH range for the determination of the logD values of basic compounds, providing an optional mild pH for IS-RPLC experiments.

Magnetic Ti3C2 MXene Nanosheets Prepared for Enrichment of Phosphopeptides.

MXenes have received lots of attention since discovered and have been applied in various fields. In this work, Ti3C2-Fe3O4 composites with exposed non-modified Ti3C2 MXene nanosheets were designed and prepared by an in situ growth strategy and then applied in the enrichment of phosphopeptides. The two-dimensional composites could interact with the phosphopeptides through a metal oxide affinity chromatography mechanism provided by Ti-O and Fe-O bonds and a hydrophilic interaction chromatography mechanism by surface hydroxyl groups. This magnetic nanomaterial with a specific surface area of 66.1 m2·g-1 had high sensitivity to phosphopeptides (0.5 nmol·L-1) and high selectivity (1:1000 of the molar ratio of ß-casein to bovine serum albumin). Non-fat milk was adopted as a real sample to preliminarily examine the applicability of the Ti3C2-Fe3O4-based protocol. Subsequently, Qingkailing injection, a kind of traditional Chinese medicine injection, was introduced to further explore the suitability of the nanocomposites for phosphopeptide enrichment from more complex matrices and satisfactory results were obtained.

Residue screening and analysis of enrofloxacin and its metabolites in real aquatic products based on ultrahigh-performance liquid chromatography coupled with high resolution mass spectrometry.

The abuse of enrofloxacin (ENR) in aquaculture and the lack of monitoring of other metabolites except ciprofloxacin (CIP) may lead to unknown harmful effects on human health. In this study, ENR metabolites were screened in real fish samples based on ultrahigh-performance liquid chromatography coupled with Q-Orbitrap mass spectrometry combined with Compound Discoverer software, and another metabolite deethylene-ENR besides CIP was detected and identified for the first time. Correspondingly, a method for the determination of ENR and CIP and the semi-quantitative analysis of deethylene-ENR in aquatic products was established. Method validation illustrated that excellent linearity and satisfactory recoveries of analytes were obtained. Limits of detection of ENR and CIP were both 0.1 µg kg-1, and their limits of quantification both 1 µg kg-1. CIP and deethylene-ENR were detected in 12 of 14 ENR-positive fish samples, so deethylene-ENR should be of concern as a possible risk candidate in aquatic products.

Determination of chlorpromazine and its metabolites in animal-derived foods using QuEChERS-based extraction, EMR-Lipid cleanup, and UHPLC-Q-Orbitrap MS analysis.

Chlorpromazine (CPZ) is abused in animal husbandry and can be extensively metabolized in humans and animals. However, the actual monitoring mainly focuses on the parent compound but lacks attention to its metabolites. A method was developed and validated firstly for identification and determination of CPZ and its four major metabolites in animal-derived foods using ultrahigh-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry in combination with QuEChERS preparation method. Satisfactory recoveries of analytes spiked in fish and pork samples ranged from 72 to 117 %, and limits of quantification were 2.0 and 1.0 µg kg-1 for fish and pork samples respectively. Moreover, through the hydrolysis experiments of CPZ, its hydrolysates, such as CPZ-sulfoxide, CPZ-N-oxide and CPZ-sulfoxide-N-oxide, were identified as potential risk compounds. The developed method has been successfully applied to the determination of CPZ and its metabolites in actual commercial samples, as well as to the screening of other CPZ-related risk compounds.

A novel particulate matter sampling and cell exposure strategy based on agar membrane for cytotoxicity study.

Laboratories use different strategies to sample and extract atmospheric particulate matter (PM), some of which can be very complicated. Due to the absence of a standard protocol, it is difficult to compare the results of PM toxicity assessment across different laboratories. Here, we proposed a novel PM sampling and cell exposure strategy based on agar membrane. The agar membrane, prepared by a simple freeze-drying method, has a relatively flat surface and porous interior. We demonstrated that the agar membrane was a reliable substitute material for PM sampling. Then the PM on the agar membranes was directly extracted with the culture medium by vortex method, and the PM on the polytetrafluoroethylene (PTFE) filters was extracted with water by the traditional ultrasonic method for comparison. The extraction efficiency was evaluated and in vitro cytotoxicity assays were carried out to investigate the toxic effects of PM extracted with two strategies on macrophage cells. The results showed that the PM extracted from agar membranes induced higher cytotoxicity and more differentially expressed proteins. Overall, the novel PM sampling-cell exposure strategy based on the agar membrane is easy to operate, biocompatible and comparable, and has low disturbance, could be an alternative sampling and extraction method for PM toxicity assessment.

A combination approach using two functionalized magnetic nanoparticles for speciation analysis of inorganic arsenic.

Mercapto- and amino-functionalized magnetic nanoparticles, Fe3O4@SiO2@MPTMS (SMNPs-MPTMS) and Fe3O4@SiO2@APTES (SMNPs-APTES), have been applied as magnetic solid-phase extraction (MSPE) sorbents to directly extract arsenite (As(III)) and arsenate (As(V)) respectively, followed by inductively coupled plasma-mass spectrometry (ICP-MS) detection. Various MSPE parameters were optimized including dose of magnetic adsorbent, pH of sample solution, loading and elution conditions of analytes, adsorption capacity and reusability of SMNPs-MPTMS and SMNPs-APTES for As(III) and As(V) respectively. Under the optimized MSPE conditions, this combined scheme possesses excellent selectivity and strong anti-interference ability without any oxidation or reduction prior to capture of these two species. It is found that with a 25-fold enrichment factor, the limits of detection of As(III) and As(V) were 23.5 and 10.5 ng L-1, respectively. To verify the reliability of the proposed protocol, a certified reference material of environmental water was analyzed, and the results for inorganic arsenic species were in close agreement with the certified values. The applicability of the combination strategy for speciation analysis of inorganic arsenic was evaluated in spiked tap, river, lake and rain water samples. Good recoveries of 89%-96% and 90%-102% were achieved for As(III) and As(V), respectively, with the relative standard deviation ranges of 3.2%-8.0% and 2.5%-7.6%. Through the characterization of functionalized magnetic nanoparticles and the optimization of MSPE experiment, it is confirmed that the existence of mercapto and amino groups on SMNPs-MPTMS and SMNPs-APTES sorbents are responsible for the extraction of As(III) and As(V), respectively, via coordination and electrostatic interactions.

Preparation of a novel amino functionalized ion-imprinted hybrid monolithic column for the selective extraction of trace copper followed by ICP-MS detection.

In this work, a novel amino functionalized Cu(II) ion-imprinted organic-inorganic hybrid monolithic column (Cu(II)-IIHMC) was prepared via one-pot co-condensation and the combination of sol-gel and ion-imprinting techniques in a fused capillary. The Cu(II)-IIHMC was used as solid phase microextraction (SPME) matrix followed by inductively coupled plasma-mass spectrometry (ICP-MS) for the analysis of trace Cu(II). The prepared Cu(II)-IIHMC has good mechanical strength, stable imprinting sites and homogeneous structure of network skeleton with large flow-through pores by optimizing the synthesis process. Under the optimized conditions, the Cu(II)-IIHMC can selectively adsorb Cu(II) with the adsorption capacity of 3.13 mg g-1. With enrichment factor of 10-fold, the calibration curve was established in the range of 0.05-50 µg L-1 with r2 of 0.9992 and the detection limit was 0.008 µg L-1 for Cu(II). Compared with the non-imprinted hybrid monolithic column (Cu(II)-NIHMC), the Cu(II)-IIHMC possesses better selectivity, anti-interference ability and adsorption capacity. The Cu(II)-IIHMC can specifically capture the target ion in the presence of competitive ions, with the selectivity coefficients exceeding 39.4. The protocol was validated by analyzing Certified Reference Materials of standard sediment, soil and iron ore, and the results were in good agreement with certified values. Moreover, the proposed in-tube SPME procedure can not only preconcentrate trace Cu(II), but also effectively reduce the matrix effect and powerfully eliminate the interference from the main metals in real samples. Therefore, the developed SPME-ICP-MS method with facile preparation, specific selectivity, high sensitivity and efficient analysis, was applied in the determination of trace Cu(II) in environmental and mineral samples with the recoveries of 89.8-111.8% in all spiked samples.

Tetrasulfonate calix[4]arene modified large pore mesoporous silica microspheres: Synthesis, characterization, and application in protein separation.

Effective protein adsorption by solid matrices from complex biological samples has attracted attention for broad application in biomedical field. Immobilization of calixarenes to solid supports is an essential process for their application in protein separation and purification. Silica is the most widely used support material in calixarene immobilization. With high concentration of polymer microspheres as templates, the large pore mesoporous silica microspheres with controllable, uniform size and structure were successfully synthesized and the resulting large pore mesoporous silica microspheres were modified with water-soluble tetrasulfonate calix[4]arene of unique hollow cavity-shaped structure. The tetrasulfonate calix[4]arene modified large pore mesoporous silica microspheres (SCLX4@LPMS) were characterized by diverse analytical techniques and their protein adsorption performance were also investigated. The obtained SCLX4@LPMS gave rise to an adsorption efficiency of >90% for cytochrome c and lysozyme within a wide pH range of 3.0-10.0 and possessed remarkably high adsorption capacity of cytochrome c (363.64 mg g-1) and lysozyme (166.11 mg g-1). The retained cytochrome c and lysozyme can be readily eluted by using phosphate buffer solution containing NaCl as a stripping reagent with the recoveries of 81% and 86% after 5 times enrichment, respectively. The SCLX4@LPMS microspheres have been applied for the selective adsorption of proteins in real samples and had the application potential in protein adsorption, drug delivery, biosensors, and other biomedical fields.

Universal gold nanoparticle modified hybrid monolithic substrate developed for facile in-column post-functionalization.

Organic-inorganic hybrid monolithic columns, due to the comprehensive advantages, have been applied as promising solid-phase separation matrices for pretreatment of complex samples in biomedical and environmental analyses, however, a tremendous time and efforts are cost to optimize the preparation methods of hybrid monolithic columns with different functional groups for various target analytes. Herein, we proposed a strategy to develop basic hybrid monolithic column materials for flexible and facile post-functionalization. Three kinds of single-functionalized (amine, thiol, and carboxyl) and two kinds of bi-functionalized (amine and thiol, and amine and carboxyl) hybrid monolithic columns were immobilized with gold nanoparticles (GNPs) as intermediary bridge to construct the universal substrates. The GNPs adsorption capacities of the five hybrid monoliths were compared through qualitative characterization and quantitative analysis. Thioglycolic acid (TGA) and an aptamer against human α-thrombin were respectively used for further functionalizing the substrates to select the most suitable hybrid monolith for optional post-functionalization. It was reported for the first time that the coverage density of TGA on functionalized monolithic column modified by GNPs was 168.41 nmol µL-1. Notably, the coverage density (2205.8 pmol µL-1) of the aptamer decorated on the hybrid monolithic column was significantly higher than most other similar materials in published works. After that, the aptamer functionalized hybrid monolithic column screened out was applied for the solid-phase microextraction of thrombin, which possessed excellent adsorption selectivity in interference experiment. Consequently, the developed GNPs modified amine- and thiol-bi-functionalized hybrid monolithic column is an attractive universal substrate to realize easy and efficient post-modification of separation materials for other target analytes in complex samples avoiding a lot of time and labor consumption in the optimization of process preparation.

Transport of environmental natural organic matter coated silver nanoparticle across cell membrane based on membrane etching treatment and inhibitors.

Environmental natural organic matters (NOMs) have great effects on the physicochemical properties of engineering nanoparticles, which may impact the transport of nanoparticles across plasma membrane and the cytotoxicity. Therefore, the kinetics, uptake pathway and mass of transporting into A549 cell membrane of silver nanoparticles (AgNPs) coated with citric acid (CA), tartaric acid (TA) and fulvic acid (FA) were investigated, respectively. CA, FA and TA enhanced the colloidal stability of AgNPs in culture medium and have greatly changed the surface plasmon resonance spectrum of AgNPs due to the absorption of CA, FA and TA on surface of AgNPs. Internalizing model showed that velocity of CA-, TA- and FA-nAg transporting into A549 cell were 5.82-, 1.69- and 0.29-fold higher than those of the control group, respectively. Intracellular mass of Ag was dependent on mass of AgNPs delivered to cell from suspension, which obeyed Logistic model and was affected by NOMs that CA- and TA-nAg showed a large promotion on intracellular mass of Ag. The lipid raft/caveolae-mediated endocytosis (LME) of A549 cell uptake of AgNPs were susceptible to CA, TA and FA that uptake of CA-, TA- and FA-nAg showed lower degree of dependent on LME than that of the control (uncoated AgNPs). Actin-involved uptake pathway and macropinocytosis would have less contribution to uptake of FA-nAg. Overall, transmembrane transport of NOMs-coated AgNPs differs greatly from that of the pristine AgNPs.

Carboxyl-functionalized hybrid monolithic column prepared by "thiol-ene" click reaction for noninvasive speciation analysis of chromium with inductively coupled plasma-mass spectrometry.

A novel carboxyl-functionalized hybrid monolithic column was developed based on "thiol-ene" click reaction via "one-pot" by choosing mercaptosuccinic acid, γ-methyl methacrylate trimethoxysilane and tetramethoxysilane as reaction monomers. The design of the hybrid monolithic column was assisted by the comparison in computational simulation with existing carboxyl-functionalized materials. The characterization by scanning electron microscopy, energy dispersive X-ray spectroscopy, N2 adsorption-desorption measurement, Fourier-transform infrared spectroscopy and elemental analysis showed that the carboxyl-functionalized material has the advantages of good permeability and high mechanical strength. Then, we used the prepared carboxyl-hybrid monolith column as solid phase microextraction adsorbent for separation of trace inorganic chromium species. Under pH 4.5, the hybrid monolith column can selectively enrich Cr(III) without adsorbing Cr(VI) and afterwards, Cr(III) can be eluted by 1.0 mol L-1 HCl. The chromium speciation separation method based on carboxyl-hybrid monolith column followed by inductively coupled plasma-mass spectrometry possessed the merits of facile preparation, low cost, simple and mild extraction condition, and sensitive detection, which has been successfully applied to the separation, enrichment and detection of inorganic chromium in environmental waters.

Insight into the hydrophilic interaction liquid chromatographic retention behaviors of hydrophilic compounds on different stationary phases.

In this work, the correlations between retention behavior and lipophilicity of a large set of hydrophilic neutral and ionic analytes were studied based on three hydrophilic interaction liquid chromatography (HILIC) stationary phases, including zwitterionic, crosslinked diol and triazole stationary phases. It was found that HILIC, due to the diversity of retention mechanism, is a more complex chromatography separation mode than reversed-phase liquid chromatography (RPLC) which has been widely accepted for lipophilicity assessment. Because electrostatic interactions contributed to the overall retention of the charged solutes on all three stationary phases, ion-strength of the mobile phase kept the same during the whole experiment. After the correlations between retention factor log k and water volume fraction Φ were investigated, the mixed retention model was revealed to be more suitable for HILIC retention behavior than other single models including partitioning and adsorption model. Moreover, in order to bridge the relationship between HILIC log k and lipophilicity parameter log D, net charge ne and Abraham solvation parameter were introduced in the quantitative structure-retention relationship (QSRR) model. Although the correlation coefficients between log D and log k were still moderate, the significant improvement in correlation has made HILIC a potential choice as the complement of RPLC for log D measurement.

In-tube solid-phase microextraction with a hybrid monolithic column for the preconcentration of ultra-trace metals prior to simultaneous determination by ICP-MS.

The preparation of an amino-functionalized hybrid monolithic column (TEOS-co-AEAPTES) via one-pot co-condensation of tetraethoxysilane (TEOS) and N-(ß-aminoethyl)-γ-aminopropyltriethoxysilane (AEAPTES) in a capillary is descibed. It was used as solid-phase microextraction (SPME) matrix followed by inductively coupled plasma-mass spectrometry (ICP-MS) for determination of trace metals. Under optimum conditions, the amino-functionalized SPME material can simultaneously retain Cu(II), Zn(II), Au(III), and Pb(II) with adsorption capacities of 148, 60, 81, and 64 µg m-1, respectively. Subsequently, these four metal ions can be quantitatively eluted using 1 mol L-1 HNO3 containing 1% thiourea. The retention mechanism of Cu(II), Zn(II), Au(III), and Pb(II) on the amino-functionalized hybrid monolith was explained as the combination of electrostatic and coordination interactions. With a 10-fold enrichment factor, the calibration curves were established in the range 0.5-100 µg L-1 with linear correlation coefficients above 0.9943 and the limits of quantitation were 0.05 µg L-1 for four target analytes. The limits of detection were 0.006, 0.012, 0.004, and 0.007 µg L-1 for Cu(II), Zn(II), Au(III), and Pb(II), respectively. The protocol was validated by analyzing Certified Reference Materials including standard sediment, soil, and nickel ore, and the results were in good agreement with their certified values. The relative standard deviations of the method were in the range 0.22-17.6%. The recoveries of the four metal ions in spiked samples were in the range 88.0-113.8%. Compared to direct ICP-MS determination, the proposed in-tube SPME procedure can effectively eliminate the interference from complex matrix, especially from those ores with very high content of main metal to improve the accuracy of analysis. Therefore the method is suitable for the simultaneous determination of ultra-trace Cu(II), Zn(II), Au(III), and Pb(II) in environmental and mineral samples. Graphical abstract The preparation of the TEOS-co-AEAPTES monolithic column and the SPME procedure of Cu(II), Zn(II), Au(III), and Pb(II).

Development of a novel amine- and carboxyl-bifunctionalized hybrid monolithic column for non-invasive speciation analysis of chromium.

A novel amine- and carboxyl-bifunctionalized organic-inorganic hybrid monolithic column (A&C-HMC) was synthesized via one-pot co-condensation of N-(2-aminoethyl)-3-aminopropyltriethoxysilane, carboxyethylsilanetriol sodium salt and tetramethoxysilane with cetyltrimethylammonium bromide and polyethylene glycol 6000 as binary porogens in this work. The introduction of the binary porogens controllably improved the morphology and pore structure of A&C-hybrid monolith (HM) and made the active sites of amine and carboxyl groups more prolific, compared with the monolith prepared with either of porogens. It is found that Cr(VI) and Cr(III) can be selectively adsorbed on A&C-HM under different pH ranges, and eluted by aqueous nitric acid solution completely. The A&C-HMC was used as needle-solid phase microextraction (SPME) matrix for direct separation and enrichment of inorganic chromium species coupled with inductively coupled plasma mass spectrometer without any oxidation/reduction treatment. Various parameters of SPME operation and analytical performance were investigated systematically, and the adsorption mechanism was also discussed and explained in depth. In view of the advantages of facile preparation, low cost, excellent speciation selectivity and high adsorption capacity to Cr(VI) and Cr(III), the A&C-HMC based SPME protocol is a promising alternative for non-disturbed speciation analysis of inorganic chromium in real environmental water samples.

Cytotoxicity and toxicoproteomic analyses of human lung epithelial cells exposed to extracts of atmospheric particulate matters on PTFE filters using acetone and water.

Differences of cytotoxicity associated with exposure to different extracts of atmospheric particulate matters (PMs) are still not well characterized by in vitro toxicoproteomics. In this study, in vitro cytotoxicity assays and toxicoproteomic analyses were carried out to investigate toxic effects of PM collected using polytetrafluoroethylene (PTFE) filters extracted with acetone for PM2.1 and water for PM2.1 and PM10 on A549 human lung epithelial cells. The cytotoxicity assays based on cell viability, cell apoptosis and reactive oxygen species generation indicated that PM2.1 extracted with acetone had the highest toxicity. iTRAQ labeling and LC-MS/MS analyses indicated that the number of differentially expressed proteins in A549 cells affected by PM2.1 extracted with acetone was noticeably higher than that of the other two groups. Hierarchical cluster analyses showed that the influences of the extracts of PM2.1 and PM10 using water on the proteome of A549 cells were similar, whereas significantly different from the effect of PM2.1 extracted with acetone. Pathways analyses indicated that PM2.1 extracted with acetone influenced the expression of proteins involved in 14 pathways including glycolysis/gluconeogenesis, pentose phosphate pathway, proteasome, etc. PM2.1 extracted with water affected the expression of proteins involved in 3 pathways including non-homologous end-joining, ribosome and endocytosis. However, PM10 extracted with water affected the expression of proteins involved in only spliceosome pathway. The extracts of PM using different extractants to detach PM from PTFE filters influenced the cytotoxic effects of PM and the proteome of A549 cells. Therefore, extractants should be assessed carefully before the investigations on cytotoxicity to improve the compatibility of experimental results among research teams.

In vitro inhalation bioaccessibility procedures for lead in PM2.5 size fraction of soil assessed and optimized by in vivo-in vitro correlation.

In order to assess and optimize frequently used in vitro inhalation bioaccessibility procedures for heavy metals in the inhalation risk assessment, in vivo inhalation bioavailability of Pb in simulated atmosphere fine particles (PM2.5) from aging soils spiked with lead compounds and field soils in lead-zinc mining areas was investigated via intranasally instilled experiments with these PM2.5 suspensions to mice and Pb bioaccessibility was extracted by using four frequently used in vitro procedures (Gamble Solution, simulated lung fluid, simulated epithelial lung fluid and artificial lysosomal fluid). Mouse exposure experiments showed that Pb was mainly distributed in the liver, kidneys, blood and spleen. Based on the kidney model, in vitro inhalation bioaccessibility of Pb extracted with optimized Gamble Solution, in which solid to liquid ratio (S/L) was optimized to 1:1000 g ml-1 and DTPA was proved to be the key effective component, showed a strong linear relationship with its in vivo inhalation bioavailability (y = 1.07x - 3.86, R2 = 0.73). Moreover, in vitro bioaccessible and bioavailable fractions of Pb were mainly from acid exchangeable and reducible fractions of Pb in PM2.5. Altogether, optimized Gamble Solution was suggested for the analysis of in vitro bioaccessibility for risk-based assessments.

Organic molecule-assisted synthesis of Fe3O4/graphene oxide nanocomposites for selective capture of low-abundance peptides and phosphopeptides.

The iron oxide nanoparticles (Fe3O4) were prepared by organic molecule-assisted method in aqueous solution. The facile synthetic process of Fe3O4 nanoparticles was conducted only by mixing FeCl2 and 2-methylimidazole (2-MIM) without any additives. A possible growth mechanism of the Fe3O4 nanocrystals was proposed for this mild reaction. Then, the Fe3O4 nanoparticles were anchored onto graphene oxide (GO) sheets in water by ultrasound-assisted method, forming an affinity probe with strong biocompatibility. Due to the hydroxy and carboxylic groups of GO sheets, Fe3O4/GO probe exhibits excellent performance for enriching low-abundance hydrophilic peptides, while the Fe3O4 nanoparticles endure the probe with specific affinity to phosphopeptides. The analytical protocol was developed for sequential enrichment of low-abundance peptides and phosphopeptides by the affinity probe. It exhibited the sequence coverage of 26% for capture of 17 low-abundance peptides from bovine serum albumin (BSA), as well as the selectivity of 1:1:100 for phosphopeptides from α-/ß-casein/BSA, and low detectable concentration of 2.5 fmol and probe reusability of 5 times for capture of phosphopeptides from α-/ß-casein. Consequently, the prepared Fe3O4/GO material possesses excellent feature as multifunctional affinity probe for low-abundance peptides including phosphopeptides from complex biological matrices detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.