Sheng-Tung Huang

Development of a long-wavelength fluorescent probe based on quinone-methide-type reaction to detect physiologically significant thiols.

We synthesized a new long-wavelength latent fluorimetric probe BCC (6) to detect physiologically significant thiols. The fluorogenic chemical transformation of BCC triggered by thiols is through a tandem reaction, thiol-induced benzoquinone reduction, and quinone-methide-type rearrangement reaction, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the near-red spectrum region with emission maxima at 595 nm, and it could be competitively inhibited by thiols scavenger, N-ethylmaleimide. The fluorescent response of BCC is insensitive to various non-thiol amino acids and biological reductants. This novel fluorimetric probe demonstrates a good relationship in detecting thiols in 1-100 microM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clinical diagnostic.

Efficient synthesis of 'redox-switched' naphthoquinone thiol-crown ethers and their biological activity evaluation

Series of naphthoquinone thiol-crown ethers had been prepared. The antibacterial, antifungal, and cytotoxic activities of these synthetic naphthoquinone thiol-crown ethers were investigated. All of the compounds tested displayed antibacterial, cytotoxic and antifungal activities. The bis-naphthoquinone thiol-crown ether 7a was the most potent inhibitor among tested analogues against Staphylococcus aureus methicillin resistance with MIC value of 2.68 microM.

Surface conjugation of zwitterionic polymers to inhibit cell adhesion and protein adsorption

Non-fouling surfaces that resist non-specific protein adsorption and cell adhesion are desired for many biomedical applications such as blood-contact devices and biosensors. Therefore, surface conjugation of anti-fouling molecules has been the focus of many studies. In this study, layer-by-layer polyelectrolyte deposition was applied to create an amine-rich platform for conjugation of zwitterionic polymers. A tri-layer polyelectrolyte (TLP) coating representing poly(ethylene imine) (PEI), poly(acrylic acid)-g-azide and PEI was deposited on various polymeric substrates via layer-by-layer deposition and then crosslinked via UV irradiation. Carboxyl-terminated poly(sulfobetaine methacrylate) p(SBMA) or poly(carboxybetaine methacrylate) p(CBMA) was then conjugated onto TLP coated substrates via a carbodiimide reaction. Our results demonstrate that the zwitterionic polymers could be easily conjugated over a wide pH range except under alkaline conditions, and almost completely block protein adsorption and the attachment of L929 cells and platelets. Therefore, this method has outstanding potential in biomedical applications that require low-fouling surfaces.

Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes

Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV-vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of -0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10(-10) mol cm(-2) and 3.36 s(-1), respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM - 2 mM with LOD of 4.1 μM, (2) 2 mM - 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.

Development of a sensitive long-wavelength fluorogenic probe for nitroreductase: A new fluorimetric indictor for analyte determination by dehydrogenase-coupled biosensors

Nitroreductase (NTR) is a flavin-containing enzyme that uses NADH as the electron source to reduce nitroaromatic compounds to the corresponding amines. Previous studies have shown that nitroreductase-targeted latent fluorophores exhibit low solubility in the aqueous media and fluoresce at lower wavelengths upon uncloaking, thus limiting their effective applications. Here, we have prepared a new switch-on long-wavelength latent fluorogenic substrate, NTRLF (4), for NTR. In the presence of NADH, NTR catalyzes the reduction of the nitroaromatic moiety in NTRLF (4), followed by the cascade reaction, 1,6-rearrangement-elimination reaction, cyclic urea formation, and concomitant ejects a long-wavelength fluorescence coumarin (8). However, this reaction was inhibited in the presence of nitroaromatic analogues. The fluorescence signal generated by the cascade reaction was specific and insensitive to various reductants. Accordingly, we propose that NTRLF and NTR in the presences of NADH constitute a useful switch-off high-throughput fluorescence sensor for screening nitroaromatic compounds. Furthermore, NTRLF in the NTR-coupled 3-hydroxybutyrate dehydrogenase and aldehyde dehydrogenase assay reactions was a sensitive fluorimetric indicator for the quantitatively measurement of 3-hydroxybutyrate and propionaldehyde, respectively within micromolar range. Our novel NTRLF and NTR-coupled dehydrogenase assay platform may thus be effectively applied for the quantitative estimation of a broad range of analytes.

Inhibitory effects of a rice hull constituent on tumor necrosis factor α, prostaglandin E2, and cyclooxygenase-2 production in lipopolysaccharide-activated mouse macrophages

Abstract: Isovitexin, isolated from rice hull of Oryza sativa, has been characterized as a potent antioxidant. Its antioxidant activity, determined on the basis of inhibition of lipid peroxidation by the Fenton reaction, was comparable with that of α‐tocopherol, a well‐established antioxidant. Isovitexin was able to reduce the amount of hydrogen peroxide production induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells. In this study, we assessed its effects on the production of tumor necrosis factor α (TNF‐α), prostaglandin E2 (PGE2), and the expression of cyclooxygenase‐2 (COX‐2) in LPS‐activated RAW 264.7 macrophages. Isovitexin inhibited the release of TNF‐α, a proinflammatory cytokine, upon LPS activation with a 50% inhibitory concentration (IC50) of 78.6 μM. Isovitexin markedly reduced LPS‐stimulated PGE2 production in a concentration‐dependent manner, with an IC50 of 80.0 μM. The expression of COX‐2 was also inhibited by isovitexin treatment. Our results suggest that suppression of ROS‐mediated COX‐2 expression by isovitexin is beneficial in reducing inflammation and carcinogenesis.

Immobilization of glucose oxidase on graphene and cobalt phthalocyanine composite and its application for the determination of glucose.

We described a simple and facile chemical reduction strategy for the preparation of graphene (GR)-cobalt phthalocyanine (CoPc) composite and explored it for the enzymatic determination of glucose. CoPc is an active mediator and electrocatalysts for the immobilization of GOx and determination of glucose. However, it is not stable on the electrode surface and also suffers from lack of conductivity. Here, we have employed GR as the suitable support to stabilize CoPc through simple chemical reduction method and the resulting composite has been used for the glucose biosensor application. Scanning electron microscopy, X-ray diffraction and Energy-dispersive X-ray spectroscopy studies confirmed the successful formation of composite. Direct electron transfer of glucose oxidase (GOx) was observed with well defined redox peaks at the formal potential of -0.44 V. The amount of electroactive GOx (Г) and electron transfer rate constant (ks) were calculated to be 3.77×10(-10) mol cm(-2) and 3.57 s(-1), respectively. The fabricated amperometric biosensor detects glucose in wide linear concentration range from 10 μM to 14.8 mM with high sensitivity of 5.0 9μA mM(-1) cm(-2). The sensor offered very low detection limit (LOD) of 1.6 μM. In addition, practical feasibility of the sensor has been explored in screen printing carbon electrode with accurate determination of glucose present in human blood serum and urine samples. Furthermore, the sensor exhibited appreciable stability, repeatability and reproducibility results.

Synthesis of a new long-wavelength latent fluorimetric indicator for analytes determination in the DT-Diaphorase coupling dehydrogenase assay system.

We synthesized a new long-wavelength latent fluorogenic probe BQC (1) to monitor DTD activity. The fluorogenic chemical transformation of BQC triggered by DTD in the presence of NADH is through a series of tandem reactions, DTD-catalyzed benzoquinone reduction, trimethyl-locks cyclization and intramolecular urea formation, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the near red spectrum region with emission maxima at 595 nm, and it could be competitively inhibited by menadione. The fluorescent response of BQC is insensitive to various biological thiol reductants. Furthermore, pro-fluorophore BQC is a sensitive fluorimetric indicator for analytes determination in the oxygen-insensitive DTD-coupled dehydrogenases assay by including NAD(+) which will convert to NADH by reaction in the presence of analytes. This novel oxygen-insensitive assay demonstrates a good relationship in detecting 3-hydroxybutyrate and glucose-1-phosphate in 1-10 microM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clinical diagnostic.

Development and biological evaluation of C60 fulleropyrrolidine-thalidomide dyad as a new anti-inflammation agent

Research studies in the field of C(60) fullerene derivatives have significantly increased due to the broad range of biological activities that were found for these compounds. We designed and prepared a new C(60) fullerene hybrid bearing thalidomide as a potential double-action anti-inflammatory agent, capable of simultaneous inhibition of LPS-induced NO and TNF-alpha production. The C(60) fulleropyrrolidine-thalidomide dyad, CLT, was an effective agent to suppress the release of NO and TNF-alpha by the LPS-stimulated macrophages RAW 264.7. Ten micromolars of CLT effectively inhibited LPS-induced NO and TNF-alpha production by 47.3+/-4.2% and 70.2+/-4% with respected to the control, respectively. Furthermore, preliminary biochemical investigation revealed that CLT was a potent agent to suppress both LPS-induced intracellular ROS production and iNOS expression, and CLT also inhibited the phosphorylation of ERK which is an important protein kinase involved in the activation of TNF-alpha synthesis in LPS-activated macrophages. We believed that the studies herein would hold promise for future development of a new generation of potent anti-inflammatory agents.

Hydrophilic Ester-Bearing Chlorogenic Acid Binds to a Novel Domain to Inhibit Xanthine Oxidase

Caffeic acid is a xanthine oxidase (XO) inhibitor that binds to the molybdopterin region of its active site. Caffeic acid phenethyl ester (CAPE) has higher hydrophobicity and exhibits stronger inhibition potency toward XO. Chlorogenic acid is a quinyl ester of caffeic acid that has increased hydrophilicity and also shows stronger XO inhibitory activity compared with caffeic acid. Caffeic acid and CAPE showed competitive inhibition against XO, whereas chlorogenic acid displayed mixed-type inhibition, implying that it binds to sites other than the active site. Structure-based molecular modeling was performed to account for the different binding characteristics of the hydrophobic and hydrophilic esters of caffeic acid. Chlorogenic acid showed weak binding to the molybdopterin region of XO, while it more strongly bound the flavin adenine dinucleotide region than it did the molybdopterin region. These results provide the basis for interactions of caffeic acid analogues with XO via various binding domains.