Ligang Hu

Determination of phenolic compounds in river water with on-line coupling bisphenol A imprinted monolithic precolumn with high performance liquid chromatography

The bisphenol A (BPA) imprinted monolithic precolumn has been prepared by in situ polymerization using 4-vinylpyridine (4-VP) and ethylene dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The column with good flow-through property was obtained by changing the molar ratio of the porogens (toluene and dodecanol). The selectivity and retention properties of the monolith for the BPA and other phenolic compounds were evaluated. The results show that the hydrophobic and hydrogen-bonding interaction plays important roles in the recognition process. The determination of BPA and other phenolic compounds with on-line solid-phase extraction (SPE) by monolithic precolumn coupled with conventional particulates packed and monolithic reversed-phase columns, respectively, was performed. The method was successfully applied to the analysis of phenolic compounds in river water.

Monitoring enzyme reaction and screening enzyme inhibitor based on MALDI-TOF-MS platform with a matrix of oxidized carbon nanotubes

A matrix assisted laser desorption/ionization time-of-flight mass spectrometry platform for quantitatively monitoring enzyme activity and screening enzyme inhibitors has been demonstrated. The described method employs a new matrix of oxidized carbon nanotubes. Compared with the traditional fluorescence approach, this label-free method has the advantage of directly identifying the substrates and products in enzymatic reactions. Moreover, the method could be conveniently carried out with any commercial mass spectrometer without modification. We quantitatively monitored the acetylcholinesterase activity and screened acetylcholinesterase inhibitors with a detection rate of about 3.3 s per sample.

Identification of metal-associated proteins in cells by using continuous-flow gel electrophoresis and inductively coupled plasma mass spectrometry.

Metals and metalloids are crucial for life and indispensable for a series of biological processes. It is estimated that a quarter to one third of all proteins require metals to carry out their functions, and roughly half of the known enzymes uses a particular metal as a cofactor. In spite of the prevalence and importance of metalloproteins, they are generally poorly characterized in many organisms. A recent study demonstrated that the microbial metalloproteome is much more extensive and diverse than we presently know. Currently, it is impossible to predict, genome-wide, the numbers and types of metals used by organisms and to define any metalloproteome until the proteins are fully characterized owing to diverse and poorly recognized metal coordination sites. Moreover, metals/metalloids have long been used for therapeutic purposes, for example, arsenic trioxide for the treatment of acute promyelocytic leukemia. The detailed molecular mechanisms, however, are still not fully understood owing to the complex functions of metals in biological systems. A robust and convenient approach, by which metals/metalloids can be mapped to their associated proteins proteome-wide is urgently needed. Such a methodology will improve our understanding of the molecular mechanisms of metal-dependent biological processes and profoundly promote metallomics research, an integrated biometal science complementary to genomics and proteomics. Gel electrophoresis has been one of the commonly used methods for separation and analysis of proteins based on their molecular mass and charge; however, it fails to provide information on metal identity and content for metalloproteins. The lack of convenient subsequent methods for specific metal detection confines its application on providing metalrelated information of corresponding proteins. Although laser ablation inductively coupled plasma mass spectrometry (LAICP-MS) and synchrotron X-ray fluorescence spectrometry (SXFS) have been used for the identification of metalbinding proteins on gels and in tissues/organs, either compromised sensitivity originating from the sample introduction system or limited accessibility to the synchrotron facility prevents their routine applications. Other strategies such as metal isotope radioautography, which employs unique metal isotopes to label metalloproteins, are also very inconvenient for daily usage. Herein, a new strategy based on column-type gel electrophoresis coupled with a metal-specific detection system, that is ICP-MS, was developed (Figure 1a), allowing both metals and their associated proteins to be examined comprehensively. Since the strategy can be used to analyze and at the same time to separate and isolate proteins, it can readily be applied to not only detect metalloproteins and/or metalbound proteins with a sensitivity at the femtomole level, but also conveniently integrate current proteomics with metallomics. We further showed the bismuth profile in cell lysates of Helicobacter pylori upon treatment with colloidal bismuth subcitrate (CBS) and further characterized metal-binding features of H. pylori SlyD (HpSlyD) inside cells. The column-type gel adopted the traditional slab gel preparation. Both native and denaturing conditions could be applied, and the gel compositions varied with the protein targets of interest. To validate the feasibility of the column gel system, three metal-binding proteins, Cu-bound bovine serum albumin (Cu-BSA), Cu,Zn superoxide dismutase (Cu,ZnSOD), and diferric transferrin (Fe2-Tf), were mixed and subjected to separation. Three bands, corresponding to Fe2Tf, Cu,Zn-SOD, and BSA, were visualized on a CoomassieBlue-stained slab gel (Figure 1b). The proteins separated by column-type gel electrophoresis gave rise to migration profiles similar to those observed in classical slab gel under comparable conditions. The elutes from the column gel system were split into two parts by using a T connection, with one for online metal measurement by ICP-MS and the other for protein identification through biological mass spectrometry analysis of the collected fractions (Figure 1a). It is noted that one peak was observed in either the Zn or Fe profile corresponding to SOD and transferrin, respectively, indicative of association of Zn ions with SOD and binding of Fe ions to Tf; whereas there are two peaks in the Cu profile, with each corresponding to a distinct molecular mass, thus suggesting that copper binds to both SOD and BSA. The amounts of metals were quantifiable (Figure S2 in [*] Dr. L. Hu, Dr. T. Cheng, Dr. B. He, Y. Wang, Y.-T. Lai, Prof. H. Sun Department of Chemistry, The University of Hong Kong Pokfulam, Hong Kong (P. R. China) E-mail: hsun@hku.hk

Rapid labeling of intracellular His-tagged proteins in living cells

Significance The hexahistidine-Ni2+-NTA system is used extensively in protein purification, and large numbers of His-tagged protein libraries exist worldwide. The application of this tagging system to image proteins in live cells would offer significant opportunities to track cellular events with minimal spatial and functional perturbation on a protein of interest. However, previously reported Ni-NTA–based probes suffer from poor membrane permeability and have been limited to label membrane proteins only. Here we report, to our knowledge, the first small fluorescent probe, Ni-NTA-AC, that can rapidly cross cell membranes to specifically target His-tagged proteins in various types of live cells, even in plant tissues. The probe will provide new opportunities for in situ analysis of various cellular events. Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Silver nanoparticles induced neurotoxicity through oxidative stress in rat cerebral astrocytes is distinct from the effects of silver ions

The rapid development of silver nanoparticles (AgNPs) based products has raised increasing concerns in view of their potential hazardous risks to the environment and human health. The roles of the released silver ions in AgNPs induced cytotoxicities are being hotly debated. Using rat cerebral astrocytes, the neurotoxicological effects of AgNPs and silver ions were investigated. Acute toxicity based on Alamar Blue assay showed that silver ions were considerably more toxic than AgNPs. Comparative studies indicated that AgNPs increased caspase activities and induced cell apoptosis under cytotoxic level of exposures, while silver ions compromised cell membrane integrity and dominantly caused cell necrosis. Cellular internalization of silver provided the basis for the cytotoxicities of these two silver species. In contrast to silver ions, intracellular reactive oxygen species (ROS) generation occurred in time- and concentration-dependent manners in astrocytes upon AgNPs stimulation, which caused subsequent c-Jun N-terminal kinases (JNK) phosphorylation and promoted the programmed cell death. Non-cytotoxic level of AgNPs exposure increased multiple cytokines secretion from the astrocytes, indicating that AgNPs were potentially involved in neuroinflammation. This effect was independent of silver ions as well. The distinct toxicological effects caused by AgNPs and silver ions provided the solid proofs for the particle-specific effects which should be concerned regarding the accurate assessment of AgNPs exposure risks.

Sulfidation as a Natural Antidote to Metallic Nanoparticles Is Overestimated: CuO Sulfidation Yields CuS Nanoparticles with Increased Toxicity in Medaka (Oryzias latipes) Embryos

Sulfidation is considered as a natural antidote to toxicity of metallic nanoparticles (NPs). The detoxification contribution from sulfidation, however, may vary depending on sulfidation mechanisms. Here we present the dissolution-precipitation instead of direct solid-state-shell mechanism to illustrate the process of CuO-NPs conversion to CuS-NPs in aqueous solutions. Accordingly, the CuS-NPs at environmentally relevant concentrations showed much stronger interference on Japanese medaka (Oryzias latipes) embryo hatching than CuO-NPs, which was probably due to elevated free copper ions released from CuS-NPs, leading to significant increase in oxidative stress and causing toxicity in embryos. The larval length was significantly reduced by CuS-NPs, however, no other obviously abnormal morphological features were identified in the hatched larvae. Co-introduction of a metal ion chelator [ethylene diamine tetraacetic acid (EDTA)] could abolish the hatching inhibition induced by CuS-NPs, indicating free copper ions released from CuS-NPs play an important role in hatching interference. This work documents for the first time that sulfidation as a natural antidote to metallic NPs is being overestimated, which has far reaching implications for risk assessment of metallic NPs in aquatic environment.

Brain-targeted distribution and high retention of silver by chronic intranasal instillation of silver nanoparticles and ions in Sprague-Dawley rats.

The wide applications of silver nanoparticles (AgNPs) have been concerned regarding their unintentional toxicities. Different exposure modes may cause distinct accumulation, retention and elimination profiles, which are closely related with their toxicities. Unlike silver accumulation profiles through other regular administration modes, the biodistribution, accumulation and elimination of AgNPs by intranasal instillation are not fully understood. This study conducted intranasal instillation of polyvinylpyrrolidone‐coated AgNPs in neonatal Sprague–Dawley rats at doses of 1 and 0.1 mg kg−1 day−1 for 4 and 12 weeks, respectively. The 4‐week recovery was also designed after the 12‐week exposure. Silver concentrations in the main tissues or organs were periodically monitored. Parallel exposures using silver ion were performed for the comparative studies. No physiological alterations were observed in AgNP exposures. In comparison, 1 mg kg−1 day−1 silver ions decreased body weight gain and caused mortality of 18.2%, showing ionic silver had a relatively higher toxicity than AgNPs. A relatively higher silver accumulation was observed in silver ion groups than AgNP groups. The silver ion release could not fully explain silver accumulation in AgNP exposures, showing silver distribution caused by particulate silver occurred in vivo. The highest silver concentration was in the liver at week 4, while it shifted to the brain after a 12‐week exposure. Dose‐related silver accumulation occurred for both AgNP and silver ion groups. The time course revealed a uniquely high concentration and retention of brain silver, implying chronic intranasal instillation caused brain‐targeted silver accumulation. These findings provided substantial evidence on the potential neuronal threat from the intranasal administration of AgNPs or silver colloid‐based products. Copyright

Isotope Tracers To Study the Environmental Fate and Bioaccumulation of Metal-Containing Engineered Nanoparticles: Techniques and Applications

The rapidly growing applicability of metal-containing engineered nanoparticles (MENPs) has made their environmental fate, biouptake, and transformation important research topics. However, considering the relatively low concentration of MENPs and the high concentration of background metals in the environment and in organisms, tracking the fate of MENPs in environment-related scenarios remains a challenge. Intrinsic labeling of MENPs with radioactive or stable isotopes is a useful tool for the highly sensitive and selective detection of MENPs in the environment and organisms, thus enabling tracing of their transformation, uptake, distribution, and clearance. In this review, we focus on radioactive/stable isotope labeling of MENPs for their environmental and biological tracing. We summarize the advantages of intrinsic radioactive/stable isotopes for MENP labeling and discuss the considerations in labeling isotope selection and preparation of labeled MENPs, as well as exposure routes and detection of labeled MENPs. In addition, current practice in the use of radioactive/stable isotope labeling of MENPs to study their environmental fate and bioaccumulation is reviewed. Future perspectives and potential applications are also discussed, including imaging techniques for radioactive- and stable-isotope-labeled MENPs, hyphenated multistable isotope tracers with speciation analysis, and isotope fractionation as a MENP tracer. It is expected that this critical review could provide the necessary background information to further advance the applications of isotope tracers to study the environmental fate and bioaccumulation of MENPs.

Arsenic toxicity in the human nerve cell line SK-N-SH in the presence of chromium and copper

As, Cr, and Cu represent one potential combination of multiple metals/metalloids exposures since these three elements are simultaneously leached from chromated copper arsenate (CCA)-treated wood, a common product used for building construction, at levels that can be potentially harmful. This study investigated the neurotoxicity of As associated with CCA-treated wood when accompanied by Cr and Cu. The toxicity was evaluated on the basis of a cytotoxicity model using human neuroblastoma cell line SK-N-SH. The cells were cultured with CCA-treated wood leachates or with solutions containing arsenate [As(V)], divalent copper [Cu(II)], trivalent chromium [Cr(III)] alone or in different combinations of the three elements. The toxicity was evaluated using variations in cell replication compared to controls after 96 h exposure. Among the three elements present in wood leachates, As played the primary role in the observed toxic effects, which were exerted through multiple pathways, including the generation of oxidative stress. DOM affected the absorption of metals/metalloids into the test cells, which however did not obviously appear to impact toxicity. As toxicity was enhanced by Cu(II) and inhibited by Cr(III) at concentrations below U.S. EPAs allowable maximum contaminant levels in drinking waters. Thus assessing As toxicity in real environments is not sufficient if based solely on the result from As.

Occurrence and speciation of polymeric chromium(III), monomeric chromium(III) and chromium(VI) in environmental samples

Laboratory experiments suggest that polymeric Cr(III) could exist in aqueous solution for a relative long period of time. However, the occurrence of polymeric Cr(III) has not been reported in environmental media due partially to the lack of method for speciating polymeric Cr. We observed an unknown Cr species during the course of study on speciation of Cr in the leachates of chromated-copper-arsenate (CCA)-treated wood. Efforts were made to identify structure of the unknown Cr species. Considering the forms of Cr existed in the CCA-treated woods, we mainly focused our efforts to determine if the unknown species were polymeric Cr(III), complex of Cr/As or complex of Cr with dissolved organic matter (DOM). In order to evaluate whether polymeric Cr(III) largely exist in wood leachates, high performance liquid chromatography coupled with inductively coupled mass spectrometry (HPLC-ICPMS was used) for simultaneous speciation of monomeric Cr(III), polymeric Cr(III), and Cr(VI). In addition to wood leachates where polymeric Cr (III) ranged from 39.1 to 67.4%, occurrence of the unknown Cr species in other environmental matrices, including surface waters, tap and waste waters, was also investigated. It was found that polymeric Cr(III) could exist in environmental samples containing μg/L level of Cr, at a level up to 60% of total Cr, suggesting that polymeric Cr(III) could significantly exist in natural environments. Failure in quantifying polymeric Cr(III) would lead to the underestimation of total Cr and bias in Cr speciation. The environmental implication of the presence of polymeric Cr(III) species in the environment deserves further study.