Intrinsically ESIPT-exhibiting and enhanced emission in polymer nanoparticles as signaling for sensing nitrite.

A straightforward approach to the fabrication of intrinsically excited-state intramolecular proton transfer (ESIPT)-fluorescent polymer nanoparticles (e-PNPs) was developed. The e-PNPs were obtained by self-assembly of the homopolymers derived from 4-aminosalicylic acid in aqueous solution. By incorporating ESIPT modules into polymer nanoparticles, the ESIPT reaction can be endowed with moderate hydrophobic micro-environment by nanoparticle scaffolds, eliciting enhanced ESIPT emission. The newly developed e-PNPs exhibit strong tautomeric fluorescence(e-FL), good photostability, low-toxicity and favourable biocompatibility in aqueous solution. Upon the addition of NO2-, the e-FL can be significantly quenched owing to the reaction of NO2- with the amide groups on e-PNPs. From this basis, the fluorescence detection of NO2- was implemented, which showed a linear relationship between 0 nM and 110 nM with a detection limit of 2.3 nM. Furthermore, e-PNPs were used as nanoprobes to monitor the NO2- levels in HeLa cells by fluorescence imaging, demonstrating the ability of discrimination from different concentrations of NO2-. The proposed method can be applied to a wide range of other ESIPT modules to integrate into polymer nanoparticles and offer highly sensitive nanosensing platform for bioanalysis and molecular imaging.

Physiological alterations of GS-CHO cells in response to adenosine monophosphate treatment.

Growth-arrested strategies (e.g. hypothermia and hyperosmolarity) have been widely employed to enhance cell-specific productivity (qP) in mammalian cell culture bioprocess. In addition to enhanced qP, alterations in cell physiology, such as cell size and cell cycle phase, have also attracted extensive attention under growth-arrested conditions. However, to date, very few reports on associations between physiological changes in growth-inhibiting approaches have been published. In this study, we explored associations between the physiological changes of GS-CHO cells in response to adenosine monophosphate (AMP) treatment. In dose response studies, AMP treatment resulted in suppressed proliferation, accumulated S-phase cells, increased cell size and enhanced qP. Subsequently, six GS-CHO clones exhibited the physiological alterations in varying degrees when treated with 7 mM AMP. But more importantly, a significant positive correlation between total intracellular protein content and mean electronic volume, an indicator of cell size (P < 0.01) was found, indicating that total intracellular protein was the determining factor in increasing cell size in this growth-arrested strategy. Besides, our results provide additional evidence that treatment with growth-arrested agents may increase cell size; the agent per se did not cause the increased productivity.

Intrinsically fluorescent and highly functionalized polymer nanoparticles as probes for the detection of zinc and pyrophosphate ions in rabbit serum samples.

Intrinsically fluorescent polymer nanoparticles (F-PNPs) were synthetized from 2-hydroxy-5-methylisophthalaldehyde and melamine by solvothermal method. F-PNPs can emit strong yellow green fluorescence at 542 nm without the conjugation to any external fluorescent agent and surface modification. Owing to the abundant amino and hydroxyl groups on their surface, the F-PNPs possess multiple binding sites, good biocompatibility and excellent water-solubility. Addition of Zn2+ to the F-PNPs solution resulted in a blue shift (Δλ=40 nm) with obvious enhancement in the fluorescence intensity at 502 nm; while there was negligible change in the presence of other metal ions. The subsequent treatment with pyrophosphate (PPi) can cause fluorescence recovery of F-PNPs by pulling the Zn2+ out of the coordination cavity of F-PNPs-Zn2+ nanocomposites. No interference was observed from other anions and nucleotides, making the F-PNPs-Zn2+ ensembles highly sensitive and selective nanoprobes for PPi. The detection limit is 2.75 × 10-8 M/L and 7.63 × 10-8 M/L for Zn2+ and PPi, respectively. The proposed nanoprobes were then used for detecting the recovery of Zn2+ and PPi in rabbit serum samples, which were found to be 99.4-104.2% and 98.6-104.7%, respectively. The present strategy for the fabrication of nanoparticles may offer a new sight for the preparation of polymer nanostructures. The F-FNPs based probes can provide an accurate method for the detection of Zn2+ and PPi in serum samples.

Highly cysteine-selective fluorescent nanoprobes based on ultrabright and directly synthesized carbon quantum dots.

Strongly green fluorescent carbon dots (CQDs) have been directly synthesized from 2,4-diaminophenylhydrazine and 2-hydroxy-5-methylisophthalaldehyde through a facile solvothermal method. The novel CQDs exhibit high fluorescence quantum yield and excellent water solubility due to the abundant amino and hydroxy groups on their surface. The use of the as-prepared CQDs combined with Cu2+ constructed a "turn-on" switch cysteine-responsive nanoprobe. In the CQDs-Cu2+ assemblies, the binding of Cu2+ to CQDs results in the fluorescence quenching of CQDs by electron transfer mechanism, while the addition of cysteine leads to the fluorescence recovery because of the competitive binding between cysteine and CQDs to Cu2+. The nanoprobes showed high sensitivity to cysteine with the detection limit of 2.6 nmol L-1. The selectivity investigation results demonstrated that the Cu2+-integrated nanoparticles were highly selective toward cysteine over the other amino acids and biologically related metal ions. The proposed nanoprobe was then employed for detecting the recovery of cysteine in rabbit serum and plasma samples and imaging the cysteine in cancer cells, and the recovery was found to be 98.2-104.0%. This "synthesis-modification integration" strategy for the fabrication of CQDs may offer a new sight for the preparation of multifunctional nanostructures and broadening the application of CQDs in bioimaging. Graphical abstract Fluorescent carbon dots (CQDs) were directly synthesized from 2,4-diaminophenylhydrazine and 2-hydroxy-5-methylisophthalaldehyde. CQDs exhibit high fluorescence quantum yield and excellent water solubility due to the abundant amino and hydroxy groups on their surface. The use of CQDs combined with Cu2+ constructed a cysteine-responsive nanoprobe, which showed high sensitivity to cysteine with the detection limit of 2.6 nM.

A specific nanoprobe for cysteine based on nitrogen-rich fluorescent quantum dots combined with Cu2.

As a new member of the carbon quantum-dot family, fluorescent nitrogen-rich quantum dots (NRQDs) were prepared by a mixed solvothermal method using 2-azidoimidazole and aqueous ammonia as reactants. These NRQDs are rich in nitrogen up to 40.2%, which are endowed with high fluorescence quantum yield, good photostability, water-solubility and favourable biocompatibility. We further explored the use of NRQDs combined with Cu2+ as a nanoprobe for sensing fluorescently of cysteine (Cys) in complex biological samples. In this sensing system, the fluorescence is significantly quenched via energy transfer from NRQDs to Cu2+ for the coordination of amino-containing groups with Cu2+. The strong affinity between Cu 2+ and Cys leads to the formation of Cu2+-Cys complexes and cause the detachment of Cu2+ from the surface of NRQDs, thus the fluorescence of NRQDs recover. This nanoprobe allows analysis of Cys by modulating the switch of the fluorescence of NRQDs with a detection limit of 5.3nM. As expected, the proposed NRQDs-Cu2+complex-based nanoprobes were successfully applied for the determination of Cys in human serum and plasma samples with recoveries ranging from 97.2% to 105.7%. The probe ensemble was also successfully applied to imaging of Cys in living cells with satisfactory results, which shows strong potential for clinical diagnosis.

The relationship of metabolic burden to productivity levels in CHO cell lines.

The growing demand for recombinant therapeutics has driven biotechnologists to develop new production strategies. One such strategy for increasing the expression of heterologous proteins has focused on enhancing cell-specific productivity through environmental perturbations. In this work, the effects of hypothermia, hyperosmolarity, high shear stress, and sodium butyrate treatment on growth and productivity were studied using three (low, medium, and high producing) CHO cell lines that differed in their specific productivities of monoclonal antibody. In all three cell lines, the inhibitory effect of these parameters on proliferation was demonstrated. Additionally, compared to the control, specific productivity was enhanced under all conditions and exhibited a consistent cell line specific pattern, with maximum increases (50-290%) in the low producer, and minimum increases (7-20%) in the high producer. Thus, the high-producing cell line was less responsive to environmental perturbations than the low-producing cell line. We hypothesize that this difference is most likely due to the bottleneck associated with a higher metabolic burden caused by higher antibody expression. Increased recombinant mRNA levels and pyruvate carboxylase activities due to low temperature and hyperosmotic stress were found to be positively associated with the metabolic burden.

[Determination of volatile organic compounds in workplace by portable gas-chromatography].

OBJECTIVE: To establish a portable gas chromatography (GC) method for the on-site rapid determination of epoxyethane, furan, dichloromethane, benzene, toluene in workplace. METHODS: By using dynamic dilutor, the volatile organic compounds (VOCs) standard gas was prepared and drawn to the capillary column of GC (PID) for separation and analysis. Based on the retention time and peak area, the VOCs was identified and quantified. RESULTS: At the conditions of 60 degrees C column temperature and 9psi pre-pressure, The five VOCs were well separated. Good linear ranges were obtained within the ranges of 0.45 - 90.72 mg/m3 for epoxyethane, 0.22 - 44.50 mg/m3 for furan, 2.19 - 437.4 mg/m3 for dichloromethane, 0.32 - 13.29 mg/m3 for benzene, 0.38 - 15.68 mg/m3 for toluene, respectively. The correlation coefficient was not less than 0.999. The detection limits were 0.03, 0.04, 0.2, 0.008 and 0.03 mg/m3, respectively. The RSD were 0.79% - 2.4%, 1.5% - 1.9%, 0.97% - 1.8%, 2.3% - 3.2% and 3.4% - 4.6%, respectively. The average recovery were 101.4% - 108.4% 85.49% - 94.98%, 97.78% - 106.2%, 99.29% - 103.5% and 95.54% - 101.7% respectively. By using the 7890A GC method, the relative tolerance for the same gas samples were 0.091% - 8.8% for epoxyethane, 3.8% - 6.7% for furan, 0.77% - 9.4% for dichloromethane, 0.24% - 1.6% for benzene, 0.31% - 8.0% for toluene. CONCLUSION: The method is portable, accurate, sensitive, rapid, and exhibits good separation and anti-interference ability. This method is suitable for the rapid detection of VOCs in workplace and also provides reference VOC detection method for the occupational health standard.

Understanding central carbon metabolism of rapidly proliferating mammalian cells based on analysis of key enzymatic activities in GS-CHO cell lines.

The central carbon metabolism (glycolysis, the pentose phosphate pathway [PPP], and the tricarboxylic acid [TCA] cycle) plays an essential role in the supply of biosynthetic precursors and energy. How the central carbon metabolism changes with the varying growth rates in the in vitro cultivation of rapidly proliferating mammalian cells, such as cancer cells and continuous cell lines for recombinant protein production, remains elusive. Based on relationships between the growth rate and the activity of seven key enzymes from six cell clones, this work reports finding an important metabolic characteristic in rapidly proliferating glutamine synthetase-Chinese hamster ovary cells. The key enzymatic activity involved in the TCA cycle that is responsible for the supply of energy became elevated as the growth rate exhibited increases, while the activity of key enzymes in metabolic pathways (glycolysis and the PPP), responsible for the supply of biosynthetic precursors, tended to decrease-suggesting that rapidly proliferating cells still depended predominantly on the TCA cycle rather than on aerobic glycolysis for their energetic demands. Meanwhile, the growth-limiting resource was most likely biosynthetic substrates rather than energy provision. In addition, the multifaceted role of glucose-6-phosphate isomerase (PGI) was confirmed, based on a significant correlation between PGI activity and the percentage of G2/M-phase cells.