Microfluidic peroxidase biochip for polyphenol synthesis

An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors.     

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum

A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum.     

C6-Like and C3-Like Molecules from the Cephalochordate, Amphioxus, Suggest a Cytolytic Complement System in Invertebrates

The mammalian immune system has cytotoxic mechanisms, both cellular and humoral, that destroy the membrane integrity of target cells. The main effector molecules of these cytolytic mechanisms—perforin, used by killer lymphocytes, and the membrane attack complex (MAC) components of the complement system—share a unique module called the MAC/perforin module. Until now, both immunological cytotoxicity and the MAC/perforin module have been reported only in jawed vertebrates. Here, we report the identification of a protein containing the MAC/perforin module from the invertebrate cephalochordate, amphioxus (Branchiostoma belcheri), using expressed sequence tag (EST) analysis of the notochord. The deduced amino acid sequence of this molecule is most similar to the primary structure of human complement component C6 and is designated AmphiC6. AmphiC6 shares a unique modular structure, including the MAC/perforin module, with human C6 and other MAC components. Another EST clone predicts the presence of a thioester-containing protein with the closest structural similarity to vertebrate C3 (therefore designated AmphiC3). AmphiC3 retains most of the functionally important residues of vertebrate C3 and is shown by phylogenetic analysis to be derived directly from the common ancestor of vertebrate C3, C4, and C5. Only opsonic activity has been assigned to the invertebrate complement system until now. Therefore, this is the first molecular evidence for complement-mediated immunological cytotoxicity in invertebrates. Received: 24 August 2001 / Accepted: 12 November 2001     

A Modular Flat Panel Photobioreactor (MFPP) for indoor mass cultivation of Nannochloropsis sp. under artificial illumination

Nannochloropsis sp. was grown in a Modular FlatPanel Photobioreactor (MFPP) consisting of sixalveolar panels each with 20.5 L culture volume and3.4 m² illuminated surface area. The panelsformed a closely-packed unit with illuminationprovided by banks of fluorescent tubes placed betweenthe panels. The whole unit was contained in athermoregulated cabinet. Continuous illumination ofone side of the panels with 115 molphoton m^-2 s^-1 attained a mean volumetricproductivity of 0.61 g (d. wt) L^-1 24 h^-1,increasing to 0.97 g (d. wt) L^-1 24 h^-1 whenthe same irradiance was provided on both sides of thepanels. With 230 mol photon m^-2 s^-1 onone side of the panel, a mean productivity of 0.85 g(d. wt) L^-1 24 h^-1 was achieved, whichreached 1.45 g (d. wt) L^-1 24 h^-1 when bothsides were illuminated. Increasing the amount of lightprovided to the culture (either by increasingirradiance or the illuminated surface area) decreasedpigment and enhanced the total fatty acid content, butdid not change significantly the content ofeicosapentaenoic acid. A MFPP of the presentdimensions could produce sufficient microalgae tosupport a hatchery producing 6 million sea breamfingerlings annually.     

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

A High Power‐Density,Mediator‐Free,Microfluidic Biophotovoltaic Device for Cyanobacterial Cells

Biophotovoltaics has emerged as a promising technology for generating renewable energy because it relies on living organisms as inexpensive, self‐repairing, and readily available catalysts to produce electricity from an abundant resource: sunlight. The efficiency of biophotovoltaic cells, however, has remained significantly lower than that achievable through synthetic materials. Here, a platform is devised to harness the large power densities afforded by miniaturized geometries. To this effect, a soft‐lithography approach is developed for the fabrication of microfluidic biophotovoltaic devices that do not require membranes or mediators. Synechocystis sp. PCC 6803 cells are injected and allowed to settle on the anode, permitting the physical proximity between cells and electrode required for mediator‐free operation. Power densities of above 100 mW m^‐2 are demonstrated for a chlorophyll concentration of 100 μM under white light, which is a high value for biophotovoltaic devices without extrinsic supply of additional energy.     

Microphysiological systems: What it takes for community adoption

Microphysiological systems (MPS) are promising in vitro tools which could substantially improve the drug development process, particularly for underserved patient populations such as those with rare diseases, neural disorders, and diseases impacting pediatric populations. Currently, one of the major goals of the National Institutes of Health MPS program, led by the National Center for Advancing Translational Sciences (NCATS), is to demonstrate the utility of this emerging technology and help support the path to community adoption. However, community adoption of MPS technology has been hindered by a variety of factors including biological and technological challenges in device creation, issues with validation and standardization of MPS technology, and potential complications related to commercialization. In this brief Minireview, we offer an NCATS perspective on what current barriers exist to MPS adoption and provide an outlook on the future path to adoption of these in vitro tools.     

Facile conversion of RNA aptamers to modular fluorescent sensors with tunable detection wavelengths

A GTP aptamer was converted to a modular fluorescent GTP sensor by conjugation of RRE (Rev responsive element) RNA and successive complex formation with a fluorophore-modified Rev peptide. Structural changes associated with substrate binding in the RNA aptamer were successfully transduced into changes in fluorescence intensity because of the modular structure of ribonucleopeptides. A simple modular strategy involving conjugation of a fluorophore-modified ribonucleopeptide to the stem region of an RNA aptamer deduced from secondary structural information helps produce fluorescent sensors, which allow tuning of excitation and detection wavelengths through the replacement of the fluorophore at the N-terminal of the Rev peptide.     

泉州市不同利用方式下土壤磷的吸附与解吸特性

分析了不同利用方式下泉州市土壤磷素吸附-解吸特征.结果表明:Langmuir等温方程式可以很好地表征土壤磷素的吸附特性;旱地和轮作地土壤对磷的吸附能力较强,而草地和林地土壤对磷的吸附能力较弱;磷的流失风险顺序为轮作地＞草地＞林地＞旱地;指导施磷量与吸附常数、最大缓冲量的大小顺序一致,为旱地＞轮作地＞林地＞草地;轮作地和草地的解吸率高于旱地和林地,土壤的缓冲能力顺序为旱地＞林地＞轮作地＞草地.主成分分析表明,平均解吸率、易解吸磷、磷吸附指数和磷零吸持平衡浓度4个指标最能反映土壤磷素流失潜力,可作为评价流失潜力的主要指标.