3D scaffold fabricated with composite material for cell culture and its derived platform for safety evaluation of drugs.

In order to overcome the weakness of conventional approaches for cell culture, and provide cells with more in vivo-like microenvironment for studying hepatotoxicity of drugs, "multiple-in-one" strategy was adopted to fabricate a 3D scaffold of silk fibroin/hydroxyapatite/poly lacticco-glycolic acid (SF/HA/PLGA), where HepG2 cells were cultivated and the toxicity of drugs to the cells was investigated. The prepared 3D scaffold proves to bear proper porosity, excellent mechanical property, steady pH environment and good biocompatibility for cell culture. Furthermore, the validity of the developed 3D-SF/HA/PLGA-scaffold based platform was verified by probing the toxicity of a known drug-induced liver injury (DILI) concern acetaminophen (APAP) to HepG2 cells. Eventually, an application of the platform to dioscin (a medicinal plant extract) reveals the hepatotoxicity of dioscin, which involves the inhibition of the expression of CYP3A4 mRNA in the cells. The developed 3D-SF/HA/PLGA-scaffold platform may become a universal avenue for safety evaluation of drugs.

Perovskite nanocrystals fluorescence nanosensor for ultrasensitive detection of trace melamine in dairy products by the manipulation of inner filter effect of gold nanoparticles.

Barium sulfate-coated CsPbBr3 perovskite nanocrystals (CsPbBr3 NCs@BaSO4) was successfully synthesized that exhibited stable and intense fluorescence property in aqueous buffer. With the CsPbBr3 NCs@BaSO4 as signal readout, an ultrasensitive fluorescence nanosensor was developed for turn-on determination of melamine by the manipulation of inner filter effect of citrate-protected gold nanoparticles (AuNPs). The fluorescence of the CsPbBr3 NCs@BaSO4 was remarkably quenched by the AuNPs due to inner filter effect. This inner filter effect could be weakened by the addition of melamine as a result of melamine-triggering aggregation of the AuNPs and subsequently led to a recovery in the fluorescence of the CsPbBr3 NCs@BaSO4. The recovery ratio was proportional to the concentration of melamine in the range of 5.0-500.0 nmol/L. The limit of detection was 0.42 nmol/L and the relative standard deviation was 4.0% for the repetitive determination of 500.0 nmol/L melamine solution (n = 11). The nanosensor was successfully applied to analysis of melamine in dairy product samples.

[Gas chromatographic determination of trace carbon monoxide and carbon dioxide in sulfur hexafluoride and its application to probing potential breakdown of high-voltage circuit breaker].

A reliable gas chromatography-flame ionization detector (GC-FID) method was developed for the accurate determination of trace CO and CO2 in high concentration of SF6. Using a combination of dual Porapak Q chromatographic columns with valve switching, SF6 was separated from the tested samples and blown out of the detection system, with the aim of ruling out the possibility of conversion column poisoning. Meanwhile, CO and CO2 were on-line separated and subsequently converted to CH4 in the presence of a nickel catalyst to increase the sensitivity for the determination of carbon-borne gases. The results showed that neither gas interfered with the determination of the other. Satisfactory linearities were achieved in the range 2-500 µL/L for both CO and CO2, with highly accurate and reproducible determination (RSD<2%). The developed GC-FID method is applicable to the determination of gases lodged in high-voltage circuit breakers toward the analysis of trace CO and CO2, thus providing a tool for probing the potential breakdown of SF6-insulated equipment.

Screening and identification of the active components from Puerariae Radix by HUVEC/CMC-LC-MS2.

Puerariae Radix (PR) serves as food and medicinal plant for thousands of years with explicit efficacy for heart diseases, while biological target specifically binding-oriented screening of the active components in PR remains a preliminary stage. Cell membrane chromatography (CMC) is newly developed approach where interactions between active components and certain biological targets can be effectively studied, Human umbilical vein endothelial cell (HUVEC) membrane, with its abundant receptors such as ß and AT1, is most eligible for constructing CMC. In this study, an HUVEC/CMC-LC-MS2 system was developed for screening active components in PR, 11 compounds were screened out and four of them were identified. Besides puerarin, the rest identified are daidzin, pueroside D and 3'-hydroxypuerarin. The study provides more reference for CMC applications and PR exploitation.

One-step maltose-functionalization of magnetic nanoparticles based on self-assembled oligopeptides for selective enrichment of glycopeptides.

Magnetic nanoparticles (MNPs) have been widely explored in enrichment of low-abundance glycoproteins/glycopeptides prior to mass spectrometry analysis in glycoproteomics. Currently, most functional groups for recognizing glycoproteins/glycopeptides are usually immobilized on the nanomaterial surface based on covalent modification, which suffers from multistep treatment, surface-dependence, and harsh conditions. In this work, we first report a facile and rapid method for surface functionalization and subsequent glycopeptides enrichment via one-step assembly of maltose-modified oligopeptides with a sequence of Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys (AEK8-maltose). In physiological conditions, AEK8-maltose readily self-organized into a complete coating layer dominated by ß-sheets on the surface of SiO2@Fe3O4 and C@Fe3O4 MNPs, which remain intact to repeat washing with acidic organic and aqueous solutions extensively used in the sample enrichment treatments. Thus, the resulting AEK8-maltose functionalized MNPs show excellent performance in enrichment of glycopeptides in standard glycoprotein digests (24 glycopeptides from horseradish peroxidase (HRP), 31 glycopeptides from immunoglobulin (IgG)) and human serum digests (282 glycopeptides), including rapid enrichment speed (5 min), high detection sensitivity (0.001 ng/µL HRP), high selectivity (mass ratios of HRP and bovine serum albumin (BSA) digests up to 1:150), good enrichment recovery (over 86.3%), remarkable stability (repeatable for more than 8 times), and excellent renewability, which are better than or comparable with the literature results reported to date. The current work based on self-assembling oligopeptides provides a mild, economic and nontoxic procedure for one-step surface functionalization of various nanomaterials.

Ratiometric fluorometric determination of mercury(II) by exploiting its quenching effect on glutathione-stabilized and tetraphenylporphyrin modified gold nanoclusters.

A ratiometric fluorometric assay for mercury(II) ion is described. It is making use of glutathione-stabilized gold nanoclusters (GSH-AuNCs) modified with tetraphenylporphyrin tetrasulfonic acid (TPPS). The resultant GSH-AuNC/TPPS nanocomposite displays dual emission (at 572 and 664 nm) under a single excitation wavelength of 365 nm. Mercury(II) ion intensively quenches the yellow fluorescence of GSH-AuNCs (peaking at 572 nm) but has a negligible effect on the red fluorescence of TPPS (at 664 nm). The ratio of fluorescence intensities at 572 and 664 nm drops linearly with Hg(II) ion concentration in the 0.02-2.0 µmol·L-1 range, and the detection limit is 7 nmol·L-1 (3sb/S). The relative standard deviation (RSD) of the assay is 2.0% at a 0.5 µmol·L-1 concentration level (n = 11). The method was successfully applied to the determination of Hg(II) ion in spiked water samples, with recoveries within the range of 87.5-107.5%. Graphical abstract Ratiometric fluorescence detection of mercury(II).

Versatile antifouling coatings based on self-assembled oligopeptides for engineering and biological materials.

The existence of nonspecific protein adsorption often results in significant challenges for microfluidic devices and laboratory cultureware used in biological experiments. Developing antifouling surfaces is thus increasingly desired by microfluidics engineers and biologists. Our previous studies have demonstrated that ionic hydrogen bonding between free ε-NH2 groups of the alkaline amino acids in proteins and surface negatively charged groups plays a critical role in strong adsorption of proteins onto the solid surfaces. Thus, the current work presents a facile and universal surface modification method based on self-assembly of oligopeptides with a sequence of Ala-Lys-Ala-Lys-Ala-Lys-Ala-Lys (AK-VIII) on poly(dimethylsiloxane) (PDMS) and polystyrene (PS) surfaces under physiological pH conditions. The results show that AK-VIII can self-organize into a compact amphipathic ß-sheet-rich coating layer on the PDMS and PS surfaces with similar coverage, which largely minimizes nonspecific adsorption of proteins. In addition, we compared the performances of BSA and AK-VIII used as blocking reagents to evaluate their inhibitory effect on nonspecific adsorption of the antigen and detection antibody in carcinoembryonic antigen (CEA) enzyme-linked immunosorbent assay (ELISA). The results showed that AK-VIII exhibits better performance than BSA for diminishing nonspecific adsorption of the antigen and detection antibody, thus providing lower background noise, a lower detection limit, and a wider linear range in CEA assays. This study provides a novel and versatile alternative for developing antifouling coatings to address nonspecific protein adsorption on both PDMS-based engineering and PS-based biological materials.

Phytochemical composition and antibacterial activity of the essential oils from different parts of sea buckthorn (Hippophae rhamnoides L.).

Essential oils from the seed, pulp, and leaf of sea buckthorn were obtained with hydrodistillation, and their phytochemical composition was analyzed through gas chromatography-mass spectrometry. Furthermore, the antibacterial activity of the oils was tested on five food-borne bacteria by spectrometry and evaluated in terms of minimum inhibitory concentration. The results indicate that the composition of all essential oils is dominated by free fatty acids, esters, and alkanes. Minimum inhibitory concentration values on each bacterium were obtained for oils from different parts. The oils from different parts exhibited nearly equal inhibitory effect on Staphylococcus aureus. The pulp oil was found to be the most effective for the rest of bacteria tested except Escherichia coli, on which seed oil shows twice the inhibitory effect to that of leaf or pulp oil. Three natural inhibitory examples were found comparable with or even better than the positive control: pulp oil on Bacillus subtilis, and pulp oil and leaf oil on Bacillus coagulans.

Study on pharmacokinetics and bioequivalence of Vonoprazan pyroglutamate in rats by liquid chromatography with tandem mass spectrometry.

Vonoprazan Fumarate (TAK-438F) is a new and effective drug approved in Japan in 2014 for treatment and prevention of acid-related diseases (ARDs), which exhibits many advantages compared with traditional proton-pump inhibitors (PPIs). However, the clinical applications of TAK0-438F suffers limitation due to the lack of injection dosage form. Efforts to overcome this limitation lead to the systhesis of Vonoprazan pyroglutamate (TAK-438P) for its high water solubility and more potent antisecretory effect. This was the first report to establish and validate a reliable and sensitive LC-MS/MS method for the quantification of TAK-438P in rat plasma and tissues (heart, liver, spleen, liver, kidney, rain, stomach and small intestine). All the features of the developed method suggested it was within bioanalytical criteria recommended by regulatory authorities. Furthermore, the developed method was applied to the exploration of the bioequivalence between TAK-438P and TAK-438F, as well as the pharmacokinetics and tissue distribution of TAK-438P. The results showed that there was no significant differences between TAK-438P and TAK-438F after oral administration of the same dose. Besides, TAK-438P was rapidly absorbed and eliminated in rat plasma. And it was widely distributed and there was no long-term accumulation in most tissues. Notably, more than 2000ng/mL was observed in stomach 12h after oral administration. The high accumulation revealed that stomach was likely to be the target organs of TAK-438P.

A visual volumetric hydrogel sensor enables quantitative and sensitive detection of copper ions.

We propose a visual volumetric sensor with 5,6-dicarboxylic fluorescein cross-linked amine-functionalized polyacrylamide hydrogel. The sensor undergoes volume response to Cu(2+) ions at the µM level, which enables naked-eye quantitative detection by reading the graduation on a pipette.

Insights into the mechanism of protein electrospray ionization from salt adduction measurements.

The mechanisms whereby protein ions are liberated from charged droplets during electrospray ionization (ESI) remain under investigation. Compact conformers electrosprayed from aqueous solution in positive ion mode likely follow the charged residue model (CRM), which envisions analyte release after solvent evaporation to dryness. The concentration of nonvolatile salts such as NaCl increases sharply within vanishing CRM droplets, promoting nonspecific pairing of Cl(-) and Na(+) with charged groups on the protein surface. For unfolded proteins, it has been proposed that ion formation occurs via the chain ejection model (CEM). During the CEM proteins are expelled from the droplet long before complete solvent evaporation has taken place. Here we examine whether salt adduction levels support the view that folded and unfolded proteins follow different ESI mechanisms. Solvent evaporation during the CEM is expected to be less extensive and, hence, the salt concentration at the point of protein release should be substantially lower than for the CRM. CEM ions should therefore exhibit lower adduction levels than CRM species. We explore the adduction behavior of several proteins that were chosen to allow comparative studies on folded and unfolded structures in the same solution. In-source activation eliminates chloride adducts via HCl release, generating protein ions that are heterogeneously charged because of sodiation and protonation. Sodiation levels measured under such conditions provide estimates of the salt adduction behavior experienced by the "nascent" analyte ions. Sodiation levels are significantly reduced for unfolded proteins, supporting the view that these species are indeed formed via the CEM.

Effects of ammonium bicarbonate on the electrospray mass spectra of proteins: evidence for bubble-induced unfolding.

Many protein investigations by electrospray ionization (ESI) mass spectrometry (MS) strive to ensure a "native" solvent environment, i.e., nondenaturing conditions up to the point of gas-phase ion formation. Ideally, these studies would employ a volatile pH buffer to mitigate changes in H(+) concentration that can occur during ESI. Ammonium acetate is a commonly used additive, despite its low buffering capacity at pH 7. Ammonium bicarbonate provides greatly improved pH stabilization, thus offering an interesting alternative. Surprisingly, protein analyses in bicarbonate at pH 7 tend to result in the formation of very high charge states, similar to those obtained when electrospraying unfolded proteins in a denaturing solvent. This effect has been reported previously (Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795), but its exact mechanistic origin remains unclear. ESI-mediated unfolding does not take place in acetate under otherwise identical conditions. We demonstrate that heating of protein-containing bicarbonate solutions results in extensive foaming, caused by CO2 outgassing. In contrast, acetate solutions do not generate foam. Protein denaturation caused by gas bubbles is a well-known phenomenon. Adsorption to the gas/liquid interface is accompanied by major conformational changes that allow the protein to act as a surfactant. The foaming of beer is a manifestation of this effect. Bubble formation in bicarbonate during ESI is facilitated by collisional and blackbody droplet heating. Our data imply that heat and bubbles act synergistically to cause unfolding during the electrospray process, while proteins reside in ESI droplets. Because of this effect we advise against the use of ammonium bicarbonate for native ESI-MS. Ammonium acetate represents a gentler droplet environment, despite its low buffering capacity.

Single-labeled hairpin probe for highly specific and sensitive detection of lead(II) based on the fluorescence quenching of deoxyguanosine and G-quartet.

Specific and homogeneous detection of heavy metal ion is of great importance for both human health care and environmental protection. We reported a highly specific and sensitive assay for fluorescent detection of Pb(2+) based on the difference in quenching ability between deoxyguanosines and G-quartet by using carboxyfluorescein-labeled hairpin DNA (F-hpDNA) as a recognition probe. In the absence of target, the fluorescence of F-hpDNA can be quenched through photoinduced electron transfer from the dye to deoxyguanosines because the formation of hairpin brings deoxyguanosines close to the FAM. In the presence of Pb(2+), the formation of G-quadruplex DNA leads to a significant decrease in fluorescence due to the effective stack of dye on the G-quartet, which obviously intensified the quenching of fluorophore. In comparison with linear DNA probe, hairpin DNA probe greatly improved the specificity, and Pb(2+) can be highly selective detected even when coexisted with other metal ions. The quenching efficiency is linear with the concentration of lead(II) over the range of 0.5-500 nM, with a limit of detection of 0.4 nM. Conformational switch from hairpin to G-quadruplex was verified by CD measurements. Moreover, the application for detection of real samples further demonstrated its reliability. Therefore, it is a selective, simple and sensitive approach for detection of lead ion, as such, it promises to provide a solid foundation for developing universal analytical method for heavy metal ions.

Study on the adsorption property of lysozyme on weak cation exchanger based on monodisperse poly(glycidymethacrylate-co-ethylenedimethacrylate) beads.

A type of weak cation exchanger was prepared based on poly(glycidylmethacrylate-co-ethylenedimethacrylate). The effects of pH and ionic strength on the adsorption behavior were studied, and the results suggested that the adsorption of lysozyme onto a weak cation exchanger is electrostatic interaction, and that the adsorption behavior is in accordance with the Langmuir adsorption model with a correlation coefficient greater than 0.99. It was also found that increasing ionic strength led to a decrease of the adsorption of lysozyme from 49.50 to 28.09 mg/g. Preliminary chromatographic experiments were conducted to test the separation properties of the weak cation exchanger, and the results demonstrated that the retention time of different proteins could be predicted in order of their isoelectric point.

Cardiac muscle sarcolemma chromatographic stationary phase and its potential application in drug screening.

A new bioactive packing material for liquid chromatography, sarcolemma chromatography stationary phase (SCSP), is presented. Its surface characteristics are investigated, and it is found that the acceptors embedded in sarcolemma remained bioactive for more than a week. The retention behavior of antagonists and activators related to cardiac muscle sarcolemma on the SCSP chromatographic column shows the screening function of the SCSP column, and the retention behavior of the active components in the aether extract from the Chinese herb Ligusticum chuanxiong Hort. on the SCSP column reveals, to a certain extent, the separation function of the SCSP column. These suggest that SCSP is a potentially useful material in drug screening.

[Study on the interactions between Ligusticum chuanxiong extract and cardiac muscle membrane receptors by CMSP chromatography].

OBJECTIVE: To study the interactions between Ligusticum chuanxiong Hort extract and cardiac muscle membrane receptors. METHOD: The cell membrane of rabbit cardiac muscle was fixed on silicon to make cell membrane stationary phase (CMSP), and then the interactions were studied by comparing the retention characteristics of the extracts from different solvents with those of the antagonists or activators corresponding to known receptors in cardiac muscle membrane, and by competition effect on the retention characteristics of extracts when adding the antagonists or activators into the mobile phase. RESULT: Water extract and ethanol extract both had retentions on CMSP; the retention characteristics of water extract could be affected when water extract was in competition with the antagonists for alpha receptor, and could not be affected when with the activator beta1 receptor. CONCLUSION: It is possible that some components in water extract may combine with alpha receptor and no component with beta1 receptor, and that some components in ethanol extract may combine with cardiac muscle cell membrane. The process between active components and receptors in vivo can be imitated through the interactions between drugs and CMSP. The method provides references for the resolution of two applications: to screen the active components from Chinese medicine, and to figure out the type of receptors involved.

[Study on the interactions between four components in Ligusticum chuanxiong rhizome and acceptors on cardiac muscle membrane].

OBJECTIVE: To study the interactions between four components in Ligusticum chuanxiong rhizome and heart cell membrane acceptors. METHOD: Through observing the retention characteristics of the four components (tetramethylpyrazine, vanillic, chrysophol, ferulic acid) on cell membrane stationary phase (CMSP) chromatographic column, whether the components combine with cell membrane acceptors was studied, and through further comparing the retention characteristics with those of known activators or antagonists corresponding to cell membrane acceptors, the kind of acceptor with which one component combines was studied. RESULT: Tetramethylpyrazine, vanillic and chrysophol had retention on CMSP chromatographic column while ferulic acid hadn't. The retention characteristics of tetramethylpyrazine were similar to activator and antagonist corresponding to a acceptor, vanillic with beta1 acceptor activator. CONCLUSION: Tetramethylpyrazine, vanillic and chrysophol can all combine with cardiac muscle membrane acceptors, while ferulic acid can not; tetramethylpyrazine probably acts on a acceptor and vanillic acts on beta1 acceptor.

Flow injection catalytic spectrophotometric simultaneous determination of nitrite and nitrate.

A new flow injection catalytic spectrophotometric method is proposed for the simultaneous determination of nitrite and nitrate based on the catalytic effect of nitrite on the redox reaction between crystal violet and potassium bromate in phosphoric acid medium and nitrate being on-line reduced to nitrite with a cadmium-coated zinc reduction column. The redox reaction is monitored spectrophotometrically by measuring the decrease in the absorbance of crystal violet at the maximum absorption wavelength of 610nm. A technique of inserting a reduction column into sampling loop is adopted and the flow injection system produces a signal with a shoulder. The height of shoulder in the ascending part of the peak corresponds to the nitrite concentration and the maximum of the peak corresponds to nitrate plus nitrite. The detection limits are 0.3ngml(-1) for nitrite and 1.0ngml(-1) for the nitrate. Up to 32 samples can be analyzed per hour with a relative standard deviation of less than 2%. The method has been successfully applied for the simultaneous determination of nitrite and nitrate in natural waters.