A facile quantification of hyaluronic acid and its crosslinking using gas-phase electrophoresis.

We report a facile, high-resolution approach to quantitatively characterize hyaluronic acid (HA) and study its crosslinking reaction using electrospray-differential mobility analysis (ES-DMA). Mobility size distributions, number concentrations, molecular mass distributions, and polydispersity index of HAs were obtained successfully via a rapid analysis by ES-DMA (< 30 min). The limit of detection, the limit of quantification, and the precision of the mobility size measurement achieve 2.5 nm, 4.0 nm, and 0.3 nm, respectively. Size exclusion chromatography (SEC) was employed as an orthogonal approach, showing that the averaged molecular mass and polydispersity index of HA measured by ES-DMA were close to the results of SEC on a semi-quantitative basis. The 1,4-butanediol diglycidyl ether (BDDE)-induced crosslinking of HA was also able to be successfully characterized through a time-dependent study using ES-DMA, which has shown the promise of direct analysis of solution-based reactions. Both the extent and the rate of HA crosslinking (induced by BDDE) were proportional to reaction temperature and concentration ratio of HA to BDDE. The activation energy of the reaction-limited BDDE-induced crosslinking of HA was found to be ≈ 21 kJ/mol. The prototype study demonstrates ES-DMA as a new method for a rapid quantitative characterization of HA and its derivative product and providing a capability of real-time monitoring of the HA crosslinking during formulation process. Graphical abstract.

Diversity of sugar acceptor of glycosyltransferase 1 from Bacillus cereus and its application for glucoside synthesis.

Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-ß-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are ß-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.

Determination of amino acids by microemulsion electrokinetic chromatography laser induced fluorescence method.

In this study, detection of 20 FITC-derivatized amino acids using an MEEKC-LIF was demonstrated. In order to achieve good separation for hydrophobic amino acids, the MEEKC method was employed and detection limits were obtained in the range of 0.32-2.2 nM, which is comparable to previous reports on amino acid analyses. Furthermore, a significant reduction in the reaction time from 1 h for conventional derivatization to 3 min for the microwave-assisted derivatization was observed and achieved, as opposed to the traditional pretreatment of real sample due to its complexity prior to the analysis of amino acid content. Finally, this microwave-assisted derivatization MEEKC-LIF method successfully determined amino acids in beverage, food, and biological samples (rat brain) with good recovery.

Ultrafast Patterning Vertically Aligned Carbon Nanotube Forest on Al Foil and Si Substrate Using Chemical Vapor Deposition (CVD).

This study introduces a method of patterning carbon nanotube (CNTs) forests that is both fast and simple. We found that, as commercially available oil-based markers undergo nanotube synthesis, a thin film forms that prevents the catalyst, ferrocene, from coming into contact with the surface of the test sample. This, thus, blocks CNT growth. Through further deduction, we used styrene maleic anhydride (SMA) to conduct CNT patterning, in addition to analyzing the relationship between the weight percent concentration of the SMA and the extent to which it blocked CNT growth. We developed two separate methods for applying ink to soft and hard substrates: one method involved ink printing and the other laser stripping. In the CNT pattern we produced, a minimum line width of around 10 µm was attained.

Quantitative determination of triglyceride by photoactivated CdSe/ZnS quantum dots through fluorescence assay.

The quantitative detection of triglycerides is an important issue for health inspection of metabolic disorders and for food and oil-refining industries. Many methods have been designed to approach this target, in which multiple reactions catalyzed by enzymes are normally coupled consecutively. In this study, we demonstrated a simple assay system containing lipase and photoactivated luminescent CdSe/ZnS quantum dots (QDs) for the quantitative detection of triglycerides. Photoactivated CdSe/ZnS QDs function as a sensitive "indicator" to reveal the minute acidity change of the assay system resulting from the enzymatic hydrolysis of triglycerides. By controlling the initial buffer condition of the assay system at 5, 10, or 20 mM phosphate buffer at pH 8.0, respectively, the quenching ratio of the QDs fluorescence intensity monitored at the maximum photoluminescence showed a linear correlation with the concentration of the examined triglyceride in the range of 0.02-6, 0.2-10, or 2-20 mM, respectively. The assay system also provides a convenient way to estimate triglyceride concentration by visualizing the color change of the QDs fluorescence. As compared to most of the existing methods, the system reported herein possessed many advantages, including simplicity, low cost, high flexibility, and high sensitivity. Furthermore, no complicated chemical modification or enzyme immobilization is needed.

A highly sensitive system for urea detection by using CdSe/ZnS core-shell quantum dots.

An original and novel assay system with urease as a catalyst and CdSe/ZnS quantum dots (QDs) as an indicator has been developed for quantitative analysis of urea. By mixing urease and QDs, the determination of urea can be performed in a quantitative manner. The detection is based on the enhancement of QD photoluminescence (PL) intensity, which is correlated to the enzymatic degradation of urea. By controlling the buffer concentration and pH, PL enhancement due to the degradation of urea is linear in the urea concentration ranging from 0.01 to 100mM. This property makes the urease/QDs system to be a promising urea-biosensing system. The newly developed system is a superior design and possesses many advantages, including its simple preparation, low cost, no enzyme immobilization required, high flexibility, and good sensitivity.

Preparation of high-performance water-soluble quantum dots for biorecognition through fluorescence resonance energy transfer.

An improved method for the synthesis of high-performance and water-soluble quantum dots (QDs) involving the encapsulation of mercaptosuccinic acid coated QDs (MSA-QDs) with poly(diallyldimethylammonium chloride) (PDDA) followed by their direct photoactivation with fluorescent radiation near 295 K to yield PDDA-coated QDs (PDDA-QDs) has been demonstrated. The quantum yield (QY) of the PDDA-QDs was significantly improved from 0.6 (QY of MSA-QDs) to 48%. By using this synthetic strategy, highly photoluminescent PDDA-QDs of varied size were readily prepared. The surface properties of PDDA-QDs and MSA-QDs were extensively characterized. The highly luminescent and positively charged PDDA-QDs serve as a useful and convenient tool for protein adsorption. With a Δ(5)-3-ketosteroid isomerase adsorbed PDDA-QD complex, the biorecognition of steroids was demonstrated through the application of fluorescent resonance energy transfer.

Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing-ß-1, 3-glucanase.

Laminaripentaose-producing ß-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of ß-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.

Water-soluble germanium nanoparticles cause necrotic cell death and the damage can be attenuated by blocking the transduction of necrotic signaling pathway.

Water-soluble germanium nanoparticles (wsGeNPs) with allyamine-conjugated surfaces were fabricated and emit blue fluorescence under ultraviolet light. The wsGeNP was physically and chemically stable at various experimental conditions. Cytotoxicity of the fabricated wsGeNP was examined. MTT assay demonstrated that wsGeNP possessed high toxicity to cells and clonogenic survival assay further indicated that this effect was not resulted from retarding cell growth. Flow cytometric analysis indicated that wsGeNP did not alter the cell cycle profile but the sub-G1 fraction was absent from treated cells. Results from DNA fragmentation and propidium iodide exclusion assays also suggested that apoptotic cell death did not occur in cells treated with wsGeNP. Addition of a necrosis inhibitor, necrostatin-1, attenuated cell damage and indicated that wsGeNP caused necrotic cell death. Cell signaling leads to necrotic death was investigated. Intracellular calcium and reactive oxygen species (ROS) levels were increased upon wsGeNP treatment. These effects can be abrogated by BAPTA-AM and N-acetyl cysteine respectively, resulting in a reduction in cell damage. In addition, wsGeNP caused a decrease in mitochondrial membrane potential (MMP) which could be recovered by cyclosporine A. The cellular signaling events revealed that wsGeNP increase the cellular calcium level which enhances the production of ROS and leads to a reduction of MMP, consequentially results in necrotic cell death.

Interplay between structure and fluidity of model lipid membranes under oxidative attack.

A proper regulation of membrane fluidity is critical for cellular activities such as communication between cells, mitosis, and endocytosis. Unsaturated lipids, a main component of biological membranes, are particularly susceptible to oxidative attack of reactive oxygen species. The oxidation of lipids can produce structural derangement of membranes and eventually alter the membrane fluidity. We have applied fluorescence correlation spectroscopy (FCS) and Raman spectroscopy to investigate the fluidity and structure of model membranes subject to oxidative attack. Hydrogen peroxide has little effect on the lateral fluidity of membranes, whereas hydroxyl radical causes a significantly increased fluidity. The latter is rationalized with the cleavage of the acyl chains of lipids caused by hydroxyl radical; this interpretation is founded on the diminished intensities of lines in Raman spectra associated with -CH(2) and C═C moieties in lipids and supported by mass-spectral measurements. The same approach provides a mechanistic account of the inhibitory capability of vitamins C and E against the increased membrane fluidity resulting from an oxidative attack. Membranes with much cholesterol exhibit a novel resistance against altered membrane fluidity induced with oxidative attack; this finding has biological implications. Our approach combining FCS and Raman measurements reveals the interplay between the structure and fluidity of membranes and provides insight into the pathophysiology of cellular oxidative injury.