A comprehensive evaluation of a polymeric zwitterionic hydrophilic monolith for nucleotide separation.

Rapid and effective separation of nucleotides (NTs) and their derivatives is crucial for studying their physiological functions. In this work, we comprehensively evaluated the separation ability of a zwitterionic hydrophilic monolith, i.e., poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine-co-N,N'-methylenebisacrylamide) (poly(SPP-co-MBA)) for NTs analysis, including its selectivity, chemical stability under extremely basic condition and compatibility with hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (HILIC-MS). The poly(SPP-co-MBA) monolith exhibited excellent chemical stability, as evidenced by the low relative standard deviation of retention time (0.16-1.05%) after 4000 consecutive injections over one month under strong alkaline elution condition (pH 10). After optimizing the separation conditions, including buffer pH and concentration, organic solvent content and column temperature, four nucleoside triphosphates, five nucleoside diphosphates and five nucleoside monophosphates were baseline separated within 7 min. Additionally, the mixtures containing one nucleoside and its corresponding mono-, di-, and triphosphates were baseline separated within only 3 min, respectively. It is good HILIC-MS compatibility was also confirmed by the satisfactory peak shape and high response of nine NTs. Overall, the proposed poly(SPP-co-MBA) monolith exhibited good mechanical stability and compatibility of HILIC-MS, making it a promising technique for NTs analysis.

Dermal Toxicity Influence of Gold Nanomaterials after Embedment in Cosmetics.

Gold nanomaterials (Au NMs) have been widely used in cosmetic products for improving the brightening, and reducing the wrinkling of, skin, etc.; however, the dermal safety of Au NMs is rarely concerned. A previous study found that cosmetics could enhance the toxicity of Au nanosheets, but different physicochemical properties of Au NMs will induce different interaction modes with ingredients of cosmetics, potentially leading to different toxicity profiles. In the present study, spherical and rodlike Au NMs were first found in commercial cosmetics, and then Au nanospheres (NSs) with different sizes and Au nanorods (NRs) with different aspect ratios were prepared to simulate these Au NMs in cosmetics and further investigate their toxicity before and after embedment in cosmetics. It was found that the primary sizes, morphologies, and optical absorptions of these Au NSs and NRs before and after embedment were similar; however, their hydrodynamic sizes and zeta potentials were noticeably different. Then, these Au NSs and NRs presented weak or no cytotoxicity against HaCaT keratinocytes, while cosmetic cream could alleviate their cytotoxicity. Moreover, the cream could enhance the accumulation of Au NSs and NRs in the skin of hairless mice, but it also alleviated the toxicological responses of Au NSs and NRs in terms of superoxide dismutase (SOD) elevation and malondialdehyde (MDA) reduction. Therefore, the embedment of Au NSs and NRs into cosmetics can alleviate the in vitro and in vivo dermal toxicities of Au NSs and NRs.

A systematic investigation of the effect of sample solvent on peak shape in nano- and microflow hydrophilic interaction liquid chromatography columns.

A frequently encountered problem in the practical application of nano- and microflow hydrophilic interaction chromatography (HILIC) columns is the distortion of peak shapes arising from a mismatch between the sample solvent and the mobile phase. An unmatched or improperly matched sample solvent can distort the peak shape of analytes and influence their retention times, thereby affecting the quality of the resulting chromatogram. In this work, the effect of sample solvent composition (mixtures of acetonitrile, water, methanol and isopropanol in different ratios) and injection volume (20-100 nL) was systematically investigated using a selection of neutral and charged compounds on a series of zwitterionic and charged small I.D. (0.1-0.3 mm) HILIC columns. For retained compounds, pure ACN was demonstrated to be the best sample solvent to obtain narrow peaks, while for compounds that eluted very close to the solvent peak, the peak shape was distorted when the sample solvent consisted of pure ACN. A highly aqueous sample solvent, which interferes with the partitioning of polar analytes into the stationary phase, was demonstrated to be detrimental for the peak shape of retained neutral compounds, while for unretained compounds that do not or hardly interact with the stationary phase, a high amount of water in the sample solvent was not problematic. For charged compounds, water in the sample solvent favored the electrostatic attraction with the stationary phase. Therefore, the retention time of charged analytes was shown to increase with increasing water content in the sample solvent. Even when a large amount of water was present in the sample solvent, the peak shapes of these compounds were still acceptable. For highly polar compounds with a limited solubility in aqueous sample solvents, it was found that a mixture of ACN and MeOH or IPA is a good alternative.

Numerical modeling and experimental testing analysis of Assembled Rubber Metal Isolator.

In order to analyze the stiffness characteristics of Assembled Rubber Metal Isolator (ARMI) more accurately, the present work elaborates on the detailed numerical modeling and analysis process of the ARMI, considering prestressing condition. By comparing the applicability of different constitutive models, the reasonable parameters of the proposed Money-Revlon constitutive model were determined by rubber compression test and least square method. Considering the structural characteristics and complex constraints of the isolator, a step-by-step analysis method is described, based on the rigid-flexible coupling theory and the contact cutting algorithm. The full Newton-Raphson algorithm is used to simulate the mechanical behavior of elastic components in ARMI, during the whole compression-torsion deformation process, while the results are verified by theoretical calculation and practical experiments, respectively. For the assembly process, the maximum relative error between numerical results and empirical formulas is 3.97%. The derived torsional curve, under the simulated pre-stress conditions, is in good agreement with the experimental results, and the maximum error is less than 8.43%. The achieved accuracy is significantly improved, compared to the existing simulation model that does not consider the pre-compression process. The proposed approach provides an effective method for the analysis of same type vibration isolator.

Separation of deamidated peptides with mixed-mode chromatography using phospholipid-functionalized monolithic stationary phases.

Deamidation of asparagine (Asn) residues of monoclonal antibodies (mAbs) plays a pivotal role in the in vivo/vitro degradation or efficacy loss of biopharmaceuticals. However, a major challenge for MS analysis of deamidation of Asn-containing peptides in mAbs, is due to the fact that there is only a 1 Da mass shift between the native form (Asn residues) and deamidated forms (n-aspartyl (n-Asp) and isoaspartyl (isoAsp) residues with identical mass). Therefore, a chromatographic separation of the deamidated proteins and/or the peptides derived therefrom is needed prior to MS analysis. In this study, the monolithic column with various stationary phases, including reverse phase (RP), single phospholipid-functionalized and mixed phospholipid-functionalized monoliths, were prepared for the separation of the deamidation-sensitive signature peptide (IYPTNGYTR) of trastuzumab and its two deamidated products, n-Asp55 residue IYPTDGYTR and isoAsp55 residue IYPTisoDGYTR. Compared to the RP monolith, the phospholipid-functionalized monoliths provided mixed-mode interactions and exhibited better peak shape and separation selectivity. The effect of the parameters, including the type and concentration of buffer, temperature and pH value on the separation performance were investigated. Under the optimal conditions, the three peptides were fully separated on a mixed phosphocholine (PC) / phosphatidic acid (PA) functionalized monolith (poly (MDPC60PA40-co-EDMA)) due to the joint contribution of hydrophobic and electrostatic interactions. Therefore, the novel method based on the mixed phospholipids-functionalized monolithic column exhibited good potential for the analysis of deamidated peptides, which will be useful for the in-depth study of post-translational modifications of mAbs.

Correction: Antimicrobial peptide based magnetic recognition elements and Au@Ag-GO SERS tags with stable internal standards: a three in one biosensor for isolation, discrimination and killing of multiple bacteria in whole blood.

[This corrects the article DOI: 10.1039/C8SC04637A.].

Self-Assembly of Au@Ag Nanoparticles on Mussel Shell To Form Large-Scale 3D Supercrystals as Natural SERS Substrates for the Detection of Pathogenic Bacteria.

Herein, we developed a natural surface-enhanced Raman scattering (SERS) substrate based on size-tunable Au@Ag nanoparticle-coated mussel shell to form large-scale three-dimensional (3D) supercrystals (up to 10 cm2) that exhibit surface-laminated structures and crossed nanoplates and nanochannels. The high content of CaCO3 in the mussel shell results in superior hydrophobicity for analyte enrichment, and the crossed nanoplates and nanochannels provided rich SERS hot spots, which together lead to high sensitivity. Finite-difference time-domain simulations showed that nanoparticles in the channels exhibit apparently a higher electromagnetic field enhancement than nanoparticles on the platelets. Thus, under optimized conditions (using Au@AgNPs with 5 nm shell thickness), highly sensitive SERS detection with a detection limit as low as 10-9 M for rhodamine 6G was obtained. Moreover, the maximum electromagnetic field enhancement of different types of 3D supercrystals shows no apparent difference, and Au@AgNPs were uniformly distributed such that reproducible SERS measurements with a 6.5% variation (613 cm-1 peak) over 20 spectra were achieved. More importantly, the as-prepared SERS substrates can be utilized for the fast discrimination of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa by discriminant analysis. This novel Au@Ag self-assembled mussel shell template holds considerable promise as low-cost, durable, sensitive, and reproducible substrates for future SERS-based biosensors.

Antimicrobial peptide based magnetic recognition elements and Au@Ag-GO SERS tags with stable internal standards: a three in one biosensor for isolation, discrimination and killing of multiple bacteria in whole blood.

In this study, a new biosensor based on a sandwich structure has been developed for the isolation and detection of multiple bacterial pathogens via magnetic separation and SERS tags. This novel assay relies on antimicrobial peptide (AMP) functionalized magnetic nanoparticles as "capturing" probes for bacteria isolation and gold coated silver decorated graphene oxide (Au@Ag-GO) nanocomposites modified with 4-mercaptophenylboronic acid (4-MPBA) as SERS tags. When different kinds of bacterial pathogens are combined with the SERS tags, the "fingerprints" of 4-MPBA show corresponding changes due to the recognition interaction between 4-MPBA and different kinds of bacterial cell wall. Compared with the label-free SERS detection of bacteria, 4-MPBA here can be used as an internal standard (IS) to correct the SERS intensities with high reproducibility, as well as a Raman signal reporter to enhance the sensitivity and amplify the differences among the bacterial "fingerprints". Thus, three bacterial pathogens (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) were successfully isolated and detected, with the lowest concentration for each of the strains detected at just 101 colony forming units per mL (CFU mL-1). According to the changes in the "fingerprints" of 4-MPBA, three bacterial strains were successfully discriminated using discriminant analysis (DA). In addition, the AMP modified Fe3O4NPs feature high antibacterial activities, and can act as antibacterial agents with low cellular toxicology in the long-term storage of blood for future safe blood transfusion applications. More importantly, this novel method can be applied in the detection of bacteria from clinical patients who are infected with bacteria. In the validation analysis, 97.3% of the real blood samples (39 patients) could be classified effectively (only one patient infected with E. coli was misclassified). The multifunctional biosensor presented here allows for the simultaneous isolation, discrimination and killing of bacteria, suggesting its high potential for clinical diagnosis and safe blood transfusions.

Preparation and evaluation of 400µm I.D. polymer-based hydrophilic interaction chromatography monolithic columns with high column efficiency.

The quest for higher column efficiency is one of the major research areas in polymer-based monolithic column fabrication. In this research, two novel polymer-based HILIC monolithic columns with 400µm I.D.×800µm O.D. were prepared based on the thermally initiated co-polymerization of N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine (SPP) and ethylene glycol dimethacrylate (EDMA) or N,N'-methylenebisacrylamide (MBA). In order to obtain a satisfactory performance in terms of column permeability, mechanical stability, efficiency and selectivity, the polymerization parameters were systematically optimized. Column efficiencies as high as 142, 000 plates/m and 120, 000 plates/m were observed for the analysis of neutral compounds at 0.6mm/s on the poly(SPP-co-MBA) and poly(SPP-co-EDMA) monoliths, respectively. Furthermore, the Van Deemter plots for thiourea on the two monoliths were compared with that on a commercial silica based ZIC-HILIC column (3.5µm, 200Å, 150mm×300µm I.D.) using ACN/H2O (90/10, v/v) as the mobile phase at room temperature. It was noticeable that the Van Deemter curves for both monoliths, particularly the poly(SPP-co-MBA) monolith, are significantly flatter than that obtained for the ZIC-HILIC column, which indicates that in spite of their larger internal diameters, they yield better overall efficiency, with less peak dispersion, across a much wider range of usable linear velocities. A clearly better separation performance was also observed for nucleobases, nucleosides, nucleotides and small peptides on the poly(SPP-co-MBA) monolith compared to the ZIC-HILIC column. It is particularly worth mentioning that these 400µm I.D. polymer-based HILIC monolithic columns exhibit enhanced mechanical strength owing to the thicker capillary wall of the fused-silica capillaries.

The effect of charged groups on hydrophilic monolithic stationary phases on their chromatographic properties.

In order to investigate the effect of charged groups present in hydrophilic monolithic stationary phases on their chromatographic properties, three charged hydrophilic monomers, i.e. N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPDA), [2-(acryloyloxy)ethyl]trimethylammonium chloride (AETA), and 3-sulfopropyl acrylate potassium salt (SPA) were co-polymerized with the crosslinker N,N'-methylenebisacrylamide (MBA), respectively. The physicochemical properties of the three resulting charged hydrophilic monolithic columns were evaluated using scanning electron microscopy, ζ-potential analysis and micro-HPLC. High column efficiency was obtained on the three monolithic columns at a linear velocity of 1mm/s using thiourea as test compound. Comparative characterization of the three charged HILIC phases was then carried out using a set of model compounds, including nucleobases, nucleosides, benzoic acid derivatives, phenols, ß-blockers and small peptides. Depending on the combination of stationary phase/mobile phase/solute, both hydrophilic interaction and other potential secondary interactions, including electrostatic interaction, hydrogen-bonding interaction, molecular shape selectivity, could contribute to the over-all retention of the analytes. Because of the strong electrostatic interaction provided by the quaternary ammonium groups in the poly (AETA-co-MBA) monolith, this cationic HILIC monolith exhibited the strongest retention for benzoic acid derivatives and small peptides with distorted peak shapes and the weakest retention for basic ß-blockers. The sulfonyl groups on the poly (SPA-co-MBA) hydrophilic monolith could provide strong electrostatic attraction and hydrogen bonding for positively charged analytes and hydrogen-donor/acceptor containing analytes, respectively. Therefore, basic drugs, nucleobases and nucleotides exhibited the strongest retention on this anionic monolith. Because of the weak but distinct cation exchange properties of the zwitterionic poly (SPDA-co-MBA) hydrophilic monolith, it exhibited the best separation for most test analytes (including phenols, ß-blockers and small peptides) in terms of selectivity, peak shape and analysis time. The poly (AETA-co-MBA) hydrophilic monolithic column provides the best separation of nucleobases and nucleosides. These results could guide the selection and application of these charged HILIC monoliths in the future.

Influence of the crosslinker type on the chromatographic properties of hydrophilic sulfoalkylbetaine-type monolithic columns.

In order to investigate the effects of the crosslinker on the separation performance of polar zwitterionic sulfoalkylbetaine-type monolithic columns, three crosslinkers, i.e. 1,4-bis(acryloyl)piperazine (PDA), ethylene dimethacrylate (EDMA) and N,N'-methylenebisacrylamide (MBA), were copolymerized with the hydrophilic monomer N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPDA). The chromatographic properties of the three hydrophilic sulfoalkylbetaine-type monolithic columns, including column efficiency, permeability, porosity and separation mechanism, were systematically compared using scanning electron microscopy or micro-HPLC. Good selectivity in micro-HPLC separations was achieved on all three monolithic columns. The results indicate that the polarity of sulfoalkylbetaine-type monolithic columns may be related to the polarity of the crosslinker, which further affects column selectivity and efficiency. A particularly high column efficiency (100,000 plates/m) was obtained on the novel poly(SPDA-co-PDA) monolithic column at a linear velocity of 1mm/s using thiourea as test analyte. A higher resolution was also observed for nucleobases, nucleosides and hydrophilic organic acids on this novel poly(SPDA-co-PDA) monolithic column compared to the other two columns.

Preparation and evaluation of a novel monolithic column containing double octadecyl chains for reverse-phase micro high performance liquid chromatography.

A novel reverse-phase monolithic stationary phase containing double long alkyl chains was prepared based on the thermally initiated co-polymerization of 3-methylacryloyl-3-oxapropyl-3-(N,N-dioctadecylcarbamoyl)-propionate (AOD) and ethylene glycol dimethacrylate (EDMA) in the presence of 2-methyl-1-propanol and 1,4-butanediol as the selected porogens. The polymerization was carefully optimized and good permeability, stability and column efficiency were observed for the final monolithic columns. The column also showed good long term stability and reproducibility. The methylene selectivity demonstrates typical reversed phase characteristics. The optimized poly (AOD-co-EDMA) monolith exhibited good selectivity for a range of non-polar test analytes such as PAHs, tocopherols and alkylphenones. A good separation of intact proteins was also observed.

Mechanism for radical cation transport in duplex DNA oligonucleotides.

We investigated the photoinduced one-electron oxidation of a series of DNA oligomers having a covalently linked anthraquinone group (AQ) and containing [(A)(n)GG](m) or [(T)(n)GG](m) segments. These oligomers have m GG steps, where m = 4 or 6, separated by (A)(n) or (T)(n) segments, where n = 1-7 for the (A)(n) set and 1-5 for the (T)(n) set. Irradiation with UV light that is absorbed by the AQ causes injection of a radical cation into the DNA. The radical cation migrates through the DNA, causing chemical reaction, primarily at GG steps, that leads to strand cleavage after piperidine treatment. The uniform, systematic structure of the DNA oligonucleotides investigated permits the numerical solution of a kinetic scheme that models these reactions. This analysis yields two rate constants, k(hop), for hopping of the radical cation from one site to adjacent sites, and k(trap), for irreversible reaction of the radical cation with H(2)O or O(2). Analysis of these findings indicates that radical cation hopping in these duplex DNA oligomers is a process that occurs on a microsecond time scale. The value of k(hop) depends on the number of base pairs in the (A)(n) and (T)(n) segments in a systematic way. We interpret these results in terms of a thermally activated adiabatic mechanism for radical cation hopping that we identify as phonon-assisted polaron hopping.

Base sequence effects in radical cation migration in duplex DNA: support for the polaron-like hopping model.

A series of anthraquinone-linked (AQ) duplex DNA oligomers were prepared and investigated. Irradiation of the AQ injects a radical cation into the DNA. The radical cation migrates through the DNA and reacts selectively at GG steps, which leads to strand cleavage after treatment with piperidine. The oligomers investigated in this work were selected to assess the effect on long-distance charge transport of placing a T base (or bases) in a strand of repeating purine bases. With notable exceptions, the amount of strand scission decreases with the distance between the AQ and the GG step. The results are consistent only with models for long-distance transport, such as thermally activated polaron-like hopping, that incorporate radical cation delocalization over two or more adjacent bases.

Nitrile biotransformations for highly efficient and enantioselective syntheses of electrophilic oxiranecarboxamides.

Catalyzed by a nitrile hydratase/amidase-containing microbial Rhodococcus sp. AJ270 whole-cell catalyst, a number of racemic trans-2,3-epoxy-3-arylpropanenitriles 1 underwent rapid and efficient hydrolysis under very mild conditions to afford 2R,3S-2-arylglycidamides 2 in excellent yield with enantiomeric excess higher than 99.5%. The overall enantioselectivity of the biotransformations originated from the combined effects of a dominantly high 2S-enantioselective amidase and low 2S-enantioselective nitrile hydratase involved in the cell. The influence of the substrates on both reaction efficiency and enantioselectivity was also discussed in terms of steric and electronic effects.