Multicellular recordings of cultured brainstem neurons in microelectrode arrays

Several vital systemic functions are controlled by the brainstem, which has been studied in a variety of experimental preparations and by various techniques, including in-vitro electrophysiological preparations. Although these in-vitro approaches have greatly advanced the understanding of brainstem neurons, most recording methods with microelectrodes and patch pipettes are invasive. To take advantage of in-vitro approaches but avoid their potential problems, we have studied brainstem neurons in microelectrode arrays (MEA). Neurons were isolated from the medulla oblongata and cultured in DMEM. Extracellular recordings were performed with no evident perturbations to the cellular environment. Neurons started firing after 24–48 h in culture, reached stable activity in 3–4 weeks, and retained this activity for at least 3 months. From their firing patterns, these neurons could be divided into tonic and bursting units. The latter could be further divided into regular and irregular bursters based on their burst intervals. Cells were stimulated or inhibited by exposure to 10% CO₂. The stimulatory effect of CO₂, though smaller, was still seen after selective ablation of serotonergic neurons or with low Ca⁺⁺ and high Mg⁺⁺ in the extracellular medium. Similar treatments had no significant effect on CO₂-inhibited units. The abundance of units with respect to their firing patterns and CO₂ responses, together with the long-term stable non-invasive recordings with no evident perturbation to cellular environments, suggests that MEA represent another promising in-vitro approach for studying brainstem neurons.This work was supported by NIH grant (HL058410, HL067890).     

Reptile freeze tolerance: metabolism and gene expression

Terrestrially hibernating reptiles that live in seasonally cold climates need effective strategies of cold hardiness to survive the winter. Use of thermally buffered hibernacula is very important but when exposure to temperatures below 0 degrees C cannot be avoided, either freeze avoidance (supercooling) or freeze tolerance strategies can be employed, sometimes by the same species depending on environmental conditions. Several reptile species display ecologically relevant freeze tolerance, surviving for extended times with 50% or more of their total body water frozen. The use of colligative cryoprotectants by reptiles is poorly developed but metabolic and enzymatic adaptations providing anoxia tolerance and antioxidant defense are important aids to freezing survival. New studies using DNA array screening are examining the role of freeze-responsive gene expression. Three categories of freeze responsive genes have been identified from recent screenings of liver and heart from freeze-exposed (5h post-nucleation at -2.5 degrees C) hatchling painted turtles, Chrysemys picta marginata. These genes encode (a) proteins involved in iron binding, (b) enzymes of antioxidant defense, and (c) serine protease inhibitors. The same genes were up-regulated by anoxia exposure (4 h of N2 gas exposure at 5 degrees C) of the hatchlings which suggests that these defenses for freeze tolerance are aimed at counteracting the injurious effects of the ischemia imposed by plasma freezing.     

Application of an enzyme chip to the microquantification of l-phenylalanine

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.     

疣粒野生稻应答黄单胞杆菌水稻致病变种（Xoo）的基因芯片

探讨疣粒野生稻应答黄单胞杆菌水稻致病变种（Xoo）的基因芯片制作,通过芯片杂交筛选抗病相关基因。芯片含有2436个片段,来自于应答撖）O的疣粒野生稻差减文库和cDNA文库,通过芯片杂交及微阵列分析基因表达,选其中800个样品点测序比对。其中,35个无同源序列,大部分有同源序列的功能未知,已知功能的序列中明显上调表达的基因有：富含脯氨酸蛋白、泛素连接酶、伸展蛋白、谷胱甘肽S-转移酶II、脂类转移酶等,明显下调表达的基因有：细胞色素P450单加氧酶、醛缩酶、金属硫蛋白、硫氧还蛋白、热激蛋白等,表达无明显变化的基因有：抗坏血酸过氧化物酶、转铜伴侣、脂酶、花丝温敏H2A蛋白等。高通量基因芯片的利用及微阵列分析是筛选抗病相关基因、获取大量抗病相关信息的有效手段。     

Field programmable chemistry: integrated chemical and electronic processing of informational molecules towards electronic chemical cells

The topic addressed is that of combining self-constructing chemical systems with electronic computation to form unconventional embedded computation systems performing complex nano-scale chemical tasks autonomously. The hybrid route to complex programmable chemistry, and ultimately to artificial cells based on novel chemistry, requires a solution of the two-way massively parallel coupling problem between digital electronics and chemical systems. We present a chemical microprocessor technology and show how it can provide a generic programmable platform for complex molecular processing tasks in Field Programmable Chemistry, including steps towards the grand challenge of constructing the first electronic chemical cells. Field programmable chemistry employs a massively parallel field of electrodes, under the control of latched voltages, which are used to modulate chemical activity. We implement such a field programmable chemistry which links to chemistry in rather generic, two-phase microfluidic channel networks that are separated into weakly coupled domains. Electric fields, produced by the high-density array of electrodes embedded in the channel floors, are used to control the transport of chemicals across the hydrodynamic barriers separating domains. In the absence of electric fields, separate microfluidic domains are essentially independent with only slow diffusional interchange of chemicals. Electronic chemical cells, based on chemical microprocessors, exploit a spatially resolved sandwich structure in which the electronic and chemical systems are locally coupled through homogeneous fine-grained actuation and sensor networks and play symmetric and complementary roles. We describe how these systems are fabricated, experimentally test their basic functionality, simulate their potential (e.g. for feed forward digital electrophoretic (FFDE) separation) and outline the application to building electronic chemical cells.     

Growth kinetics of microalgae in microfluidic static droplet arrays

We investigated growth kinetics of microalgae, Chlorella vulgaris, in immobilized arrays of nanoliter‐scale microfluidic drops. These static drop arrays enabled simultaneous monitoring of growth of single as well as multiple cells encapsulated in individual droplets. To monitor the growth, individual drop volumes were kept nearly intact for more than a month by controlling the permeation of water in and out of the microfluidic device. The kinetic growth parameters were quantified by counting the increase in the number of cells in each drop over time. In addition to determining the kinetic parameters, the cell‐size distribution of the microalgae was correlated with different stages of the growth. The single‐cell growth kinetics of C. vulgaris showed significant heterogeneity. The specific growth rate ranged from 0.55 to 1.52 day^?1 for different single cells grown in the same microfluidic device. In comparison, the specific growth rate in bulk‐scale experiment was 1.12 day^?1. It was found that the average cell size changes significantly at different stages of the cell growth. The mean cell‐size increased from 5.99 ± 1.08 to 7.33 ± 1.3 µm from exponential to stationary growth phase. In particular, when multiple cells are grown in individual drops, we find that in the stationary growth phase, the cell size increases with the age of cell suggesting enhanced accumulation of fatty acids in older cells. Biotechnol. Bioeng. 2012; 109: 2987–2996. © 2012 Wiley Periodicals, Inc.     

Chip-based protein-protein interaction studied by atomic force microscopy

In this article, a technique for accurate direct measurement of protein‐to‐protein interactions before and after the introduction of a drug candidate is developed using atomic force microscopy (AFM). The method is applied to known immunosuppressant drug candidate Echinacea purpurea derived cynarin. T‐cell/CD28 is on‐chip immobilized and B‐cell/CD80 is immobilized on an AFM tip. The difference in unbinding force between these two proteins before and after the introduction of cynarin is measured. The method is described in detail including determination of the loading rates, maximum probability of bindings, and average unbinding forces. At an AFM loading rate of 1.44 × 10⁴ pN/s, binding events were largely reduced from 61 ± 5% to 47 ± 6% after cynarin introduction. Similarly, maximum probability of bindings reduced from 70% to 35% with a blocking effect of about 35% for a fixed contact time of 0.5 s or greater. Furthermore, average unbinding forces were reduced from 61.4 to 38.9 pN with a blocking effect of ～37% as compared with ～9% by SPR. AFM, which can provide accurate quantitative measures, is shown to be a good method for drug screening. The method could be applied to a wider variety of drug candidates with advances in bio‐chip technology and a more comprehensive AFM database of protein‐to‐protein interactions. Biotechnol. Bioeng. 2012; 109: 2460–2467. © 2012 Wiley Periodicals, Inc.     

Optical Absorption Enhancement in Freestanding GaAs Thin Film Nanopyramid Arrays

Although III–V compound semiconductor multi‐junction cells show the highest efficiency among all types of solar cells, their cost is quite high due to expensive substrates, long epitaxial growth and complex balance of system components. To reduce the cost, ultra‐thin films with advanced light management are desired. Here effective light trapping in freestanding thin film nanopyramid arrays is demonstrated and multiple‐times light path enhancement is realized, where only 160 nm thick GaAs with nanopyramid structures is equivalent to a 1 μm thick planar film. The GaAs nanopyramids are fabricated using a combination of nanosphere lithography, nanopyramid metal organic chemical vapor deposition (MOCVD) growth, and gas‐phase substrate removal processes. Excellent optical absorption is demonstrated over a broad range of wavelengths, at various incident angles and at large‐curvature bending. Compared to an equally thick planar control film, the overall number of photons absorbed is increased by about 100% at various incident angles due to significant antireflection and light trapping effects. By implementing these nanopyramid structures, III–V material usage and deposition time can be significantly reduced to produce high‐efficiency, low‐cost thin film III–V solar cells.     

微电极阵列芯片技术研究48h房颤犬左、右心耳电生理特性

目的应用微电极阵列芯片（microelectrode arrays chip,MEA）技术评价48 h房颤（atrial fibrillation,AF）犬左、右心耳（LAA、RAA）的电生理特性。方法随意来源犬12只,以600次/分起搏右心房建立AF模型,分为48 h AF组（n=6）和对照组（n=6）。造模成功后迅速开胸剪取LAA、RAA,置于盛有台式液的MEA中,分别记录AF组及对照组LAA、RAA场电位（field action potential,FAP）形态、振幅、放电频率及激动传导情况。结果 AF组LAA、RAA组织FAP节律绝对不齐,LAA（185.22±25.62）次/分,较对照组（156.44±8.88）次/分增加15.67%（P〈0.01）,RAA（102.39±16）次/分,较对照组（156.44±8.88）次/分减慢34.62%（P〈0.01）。48 h AF组LAA组织电压（458.33±26.73）μV较对照组（740.55±18.93）μV降低38.11%（P〈0.01）,RAA（504.83±39.93）μV较对照组（840.56±18.93）μV明显降低（P〈0.01）,48 h房颤组LAA组织FAP时程（45.28±8.59）ms较对照组（70.77±6.98）ms缩短15 ms（P〈0.01）。RAA（61.78±7.1）ms较对照组（75.83±7.63）ms缩短14 ms（P〈0.01）。48 h AF组LAA、RAA FAP传导异质性增加。结论应用MEA技术可反映心肌组织片场电位电生理特性,48 h AF后LAA放电频率增加,频率绝对不齐,LAA、RAA电压降低,场电位时程延长。