这项工作是支持的授予国家卫生研究院，美国国立卫生研究院/ NCRR号P41 - RR 016292（ACERT）。

1。材料在这项工作中使用的所有溶剂，试剂级。自旋陷阱，包括N -叔丁基-α- phenylnitrone（PBN）和标准的自旋标签2,2,6,6 - 四甲基- 1 - piperinyoxyl（TEMPO）购自Sigma公司，并使用所提供的。植物抗氧化剂获得商业斯旺森，美国公司。 2。香烟烟雾中的制备和自由基的分析<ol><li>要引入过滤器的抗氧化剂，抗氧化剂碧萝芷和葡萄籽提取物首先被溶解在95％的乙醇，而番茄红素溶于丙酮。溶剂抗氧化剂的溶解度不同而有所不同。 </li><li>抗氧化剂的量为0.4毫克/过滤器使用。然后，他们被涂有10毫克的活性炭。为此，活性炭，搅拌〜12小时过滤和真空干燥，在厌氧条件下的抗氧化解决方案。 </l我&gt; <li>抗氧化剂，然后引入到传统的醋酸纤维过滤器（CA过滤器）。为此，过滤器被切割成两块。涂植物抗氧化剂之间插入两片过滤器，并用一块胶带包裹，形成一个过滤器 - 过滤器抗氧化剂的三明治（图1A）。后来这个组合过滤器连接到含有烟草的卷烟棒。过滤器除了没有抗氧化剂的抗氧化过滤器相同的方式控制。 </li><li>前吸烟模拟，研究香烟的解，并保持在一个恒定的湿度环境中使用2天的最低饱和溴化钠溶液（20℃，相对湿度60％）。 </li><li>常规分析吸烟模拟使用一个单端口的吸烟装置，如图1所示，在室温下进行，由水抽吸或GAST真空泵DOA - P104 - BN /空气压缩机（Benton Harbor的，MICH）连接自旋捕获大会通过的一端开一个丁字路口。泡芙被堵塞的开口端保持开放之间喷。检查流量计和气体流量设置2.2〜SCFH = 17.5毫升/ S通过调整泵和发泄之间放置一个阀。 </li><li>噗量35毫升的条件下，每隔60秒，类似1 2秒的时间反复研究香烟被熏。吸烟气相自由基的定量估计的模拟图所示的自旋捕集系统的结合。 1。 </li><li>每个卷烟激烈的喷10（35毫升/粉扑）。气相自由基的MCS通过剑桥过滤垫，然后引入到自旋捕集溶液（0.05 mol PBN的苯，2.0毫升）。 </li><li>最后粉扑后，冒泡诱捕的解决办法是重新调整其初始体积相同的苯（2.0毫升）。一个等份转入了〜250毫米长，3 mm内径的玻璃管一端密封。 </li><li>捕获的解决方案是使用一个冷冻泵解冻过程无氧。它被冷冻在液氮下，真空应用。接着是氩气气氛下解冻，让被困气泡逃脱，并再次冻结。管是这个周期重复三次后，火焰在真空状态下密封，并在进一步的ESR测量。这一步是必需的，因为氧气，这是很易溶于苯，拓宽有机自由基的ESR线。脱氧显着提高信号的信噪比。 </li><li>在标准条件下的9.34 GHz的频率为X波段的ESR光谱在Bruker EMX谱仪上记录。在大多数实验中使用的光谱仪设置为:中心领域3312.5G，扫描宽度80G，0.5G调制幅度，时间常数82微秒，扫描时间40秒。 </li><li>自旋陷阱加合物相对稳定的实验条件下，在某些情况下REQuired 25的积累，这通常〜20分钟。然而，12小时后在苯溶液中的ESR信号强度下降〜5倍。 </li><li>为了量化诱捕加合物的浓度，其最初的一阶导数的ESR谱进行了整合。由此产生的吸收光谱显示了广阔的单背景下，最有可能是由于烟雾中的烟尘/焦油产品。 后加减此背景下，诱捕加合物的分离三重集成了一个更多的时间（图2）。 </li></ol> 3。代表性的成果在燃烧香烟产生的烟雾（气相）的自由基，大部分是瞬时和不稳定。为了观察这些自由基自旋捕集技术是就业。它能够捕捉到他们转化为一个更稳定的自旋加合物ESR（图1）可以通过检测气相自由基。在本研究中，0.05自旋捕获解决方案中号PBN的是用来收集烟气相自由基，这是一个氧和碳中心自由基，这是难以分开 15的混合物。然而，在我们的案例中，所观察到的超精细分裂常数为 N = 13.7G和一个 H = 1.95G捕获的烷氧自由基（RO.）2加合物，这说明它们的主要产品相应的值非常相似。我们发现，ESR信号弱和低的重复性，在我们的初步测量观察（图2）由于烟气流动的水分。要解决这个问题，我们之间的剑桥过滤和自旋捕集溶液液氮（LN2）陷阱。从2号法律公告陷阱速冻MCS流玻璃管内壁上的水和捕捉。这大大提高ESR信号，并允许高度可重复性的结果（图3）。 被困的自由基的数量确定使用参考样本。对于对照样品无抗氧化剂，双积分频谱的比较与节奏的已知浓度的组成双频谱估计典型的加合物在苯浓度为1.24μM（图2）。由于通过每根香烟在吸烟通过的空气量是350毫升，这给自由〜7.1X10 -9中号的MCS气相自由基浓度的估计，和一个自由基的总数从气相被困整个卷烟的15〜1.5X10。总额估计在整个香烟的烟雾，包括气体和颗粒相的自由基，〜10月16日 9自由基。 观察了不同层次的植物抗氧化剂干流香烟烟雾气相自由基的清除作用。他们的清除率分别为图。 4。番茄红素和葡萄籽提取物显示率最高，而低ER率为碧萝芷（图4）。 <img alt="图1" src="/files/ftp_upload/3406/3406fig1.jpg" /> 图1。改善吸烟收集卷烟主流烟雾（MCS）使用自旋捕集气相自由基的仿真设计图。 MCS是制定水抽吸通过CA过滤器，然后剑桥过滤器（黄色滤波器），并通过液氮陷阱传递给删除H 2 O气相产品终于自旋陷阱，并通过自旋捕集溶液起泡。植物抗氧化剂之间放置两片常规的醋酸纤维过滤器（扩大）在圆一个香烟清除自由基在MCS。 <img alt="图2" src="/files/ftp_upload/3406/3406fig2.jpg" /> 图2。自旋捕获加合物在苯浓度的定量估计，需要减去一个广阔的背景信号从第一INTEGRA升谱。自旋捕获加合物的超精细分裂参数为H = 1.95 克，N = 13.7G。 <img alt="图3" src="/files/ftp_upload/3406/3406fig3.jpg" /> 图3通过液氮陷阱的显着提高的MCS自旋捕获获得的ESR信号的质量。 <img alt="图4" src="/files/ftp_upload/3406/3406fig4.jpg" /> 图4。天然抗氧化剂在MCS自由基浓度的影响。相对信号强度:控制 - 100％，碧萝芷 - 55％，葡萄籽提取物 - 12％，番茄红素 - 10％。

<ol>
	<li>Baum, S. L., Anderson, I. G. M., Baker, R. R., Murphy, D. M., Rowlands, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+spin+resonance+and+spin+trap+investigation+of+free+radicals+in+cigarette+smoke:+development+of+a+quantification+procedure.">Electron spin resonance and spin trap investigation of free radicals in cigarette smoke: development of a quantification procedure.</a> Analytica Chimica. Acta. 481, 1-13 (2003).</li><li>Bluhm, A. L., Weinstein, J., Sousa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+tobacco+smoke.">Free radicals in tobacco smoke.</a> Nature. 229, 500-500 (1971).</li><li>Church, D. F., Pryor, W. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+carcinogens+and+lung+cancer.">Tobacco smoke carcinogens and lung cancer.</a> Journal of the National Cancer Institute. 91, 1194-1210 (1999).</li><li>Kodama, M., Kaneko, M., Aida, M., Inoue, F., Nakayama, T., Akimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radical+chemistry+of+cigarette+smoke+and+its+implication+in+human+cancer.">Free radical chemistry of cigarette smoke and its implication in human cancer.</a> Anticancer. Res. 17, 433-437 (1997).</li><li>Nair, A. K., Brandt, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+smoking+on+health+care+costs.">Effects of smoking on health care costs.</a> Journal of Oklahoma State Medical Association. 93, 245-250 (2000).</li><li>Nishizawa, M., Kohno, M., Nishimura, M., Kitagawa, A., Niwano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+Peroxyradicals+in+Cigarette+Smoke+and+the+Scavenging+Effect+of+Shikonin,+a+Naphthoquinone+Pigment.">Presence of Peroxyradicals in Cigarette Smoke and the Scavenging Effect of Shikonin, a Naphthoquinone Pigment.</a> Chem. Pharm. Bull. 53, 796-799 (2005).</li><li>Pryor, W. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+extraction+of+lycopene+from+tomato+processing+byproducts.">Supercritical fluid extraction of lycopene from tomato processing byproducts.</a> J. Agric. Food. Chem. 50, 2638-2643 (2002).</li><li>Shopland, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+use+and+its+contribution+to+early+cancer+mortality+with+a+special+emphesis+on+cigarette+smoking.">Tobacco use and its contribution to early cancer mortality with a special emphesis on cigarette smoking.</a> Environmental. Health. Perspectives. 103, 131-142 (1995).</li><li>Shrikhandle, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wine+products+with+health+benefits.">Wine products with health benefits.</a> Food Res. Int. 33, 469-474 (2000).</li><li>Valavanidis, A., Haralambous, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+by+electron+paramagnetic+resonance+of+free+radical+species+in+the+mainstream+and+sidestream+smoke+of+cigarettes+with+conventional+acetate+filters+and+'bio-filters'.">A comparative study by electron paramagnetic resonance of free radical species in the mainstream and sidestream smoke of cigarettes with conventional acetate filters and 'bio-filters'.</a> Redox. 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没有利益冲突的声明。

一个可靠的估计不同的烟草烟雾中的自由基清道夫的作用，需要一个自由基的定量检测的重复性技术。此前1，它已被证明，小体积浓度较高自旋陷阱的解决方案，在非极性溶剂被困免受烟草烟雾危害的自由基最有效的。香烟烟雾中始终包含水蒸气从燃烧的有机化合物和烟草中的残留水分，可以在诱捕溶剂。这在PBN自旋捕集溶液中的掺加水，大大降低了自旋捕集加合物的寿命和其ESR信号强度。通过简单的通过用液氮冷却的U形管的MCS​​取出水分显着提高ESR谱的质量，在我们的实验中，即使一些气相自由基的一小部分，也可能会陷入冰冻的表面上。 采用t他的技术，我们比较不同的自由基清除引入到香烟过滤嘴的天然化合物的相对效率。我们发现，番茄红素和葡萄籽提取物合并成香烟过滤嘴后，立即能够清除90％的自由基气相MSC。如此高的清除能力跻身最有效的报告，如血红蛋白和shikonine 8,14激进保护这些廉价的现成的天然化合物。然而，失去了我们的实验与研究的天然抗氧化剂的卷烟装载过滤器清除一周后在室温下的存储能力的一个显着的一部分。要解决这个问题，可以刺激商业香烟过滤嘴中的番茄红素和葡萄籽提取物未来的应用。

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

吸烟是与人类癌症有关。据报道，大多数的肺癌死亡是由抽烟5,6,7,12引起的。虽然烟草在香烟烟雾中的颗粒相的焦油和相关产品致癌，致突变有关的疾病的主要成因，香烟烟雾中含有大量自由基，也作为一个重要的致癌物质9,10组的考虑。自由基攻击破坏蛋白质的结构，脂类和DNA序列的细胞成分，并增加发展各类癌症的风险。吸入的自由基会产生许多的烟草烟雾在肺3对健康的负面影响，有助于加合物。已进行研究，以减少香烟烟​​雾中的自由基，减少吸烟引起的损伤风险。据报道，血红蛋白和血红素的化合物可部分清除一氧化氮，无功氧化剂和4香烟烟雾致癌的挥发性nitrosocompounds 。一个"生物过滤器"组成的血红蛋白和活性炭是用于清除自由基和消除香烟烟雾14至90％的自由基。然而，由于成本的无效，它有没有被成功地商业化。另一项研究表明紫草，中国草药8的一个组成部分良好的清除效率。在本研究中，我们报告一个协议，用于香烟过滤嘴引入气相自由基清除香烟烟雾和卷烟主流烟气气相自由基的清除效果（MCS）的测量，使用自旋捕获常见的天然抗氧化剂提取物电子自旋共振（ESR）光谱1,2,14。我们表现​​出较高的番茄红素和葡萄籽提取物可能指向未来应用在香烟过滤嘴的清除能力。这些利弊的一个重要优势pective拾荒者，他们可以在获得大量番茄或葡萄酒行业的副产品分别为 11,13

Watch this Scientific Journal Video about A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke at JoVE.com

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

自旋捕获ESR波谱被用来研究的植物抗氧化剂番茄红素，碧萝芷和葡萄籽提取物对香烟烟雾中的自由基清除气相的作用。

卷烟主流烟气中检测和清除气相自由基议定书

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

a-protocol-for-detecting-scavenging-gas-phase-free-radicals

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

19.3K 次观看.  CDCF-AOX Lab. 自旋捕获ESR波谱被用来研究的植物抗氧化剂番茄红素，碧萝芷和葡萄籽提取物对香烟烟雾中的自由基清除气相的作用。

视频: A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

细胞色素封装<em&gt;ç</em&gt;在二氧化硅气凝胶Nanoarchitectures没有金属纳米粒子，同时保持气相生物活性

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...