作者感谢杜兰大学细胞和分子生物学系的 Dr. 罗伯特. Dotson 协助编辑杜兰大学化学系的手稿和 Dr. 詹姆斯. 布林协助采用质谱法设计。作者进一步承认杜兰大学细胞和分子生物学系和杜兰大学化学系的支持和使用空间和设备。这项工作得到了烟草产品管理科学研究金和杜兰大学科学与工程学院安德森的支持。

1. 装置的装配
<ol><li>将100毫升傅书礼烧瓶 (图 1, #4) 固定在钢制圆环架上, 并通过填充50克氯化钙来形成一个真空阱, 作为干燥剂。用橡胶通孔塞子密封烧瓶, 用石蜡膜包裹塞子接合处, 并在孔中运行吸管。</li>
	<li>使用乙烯基油管, 将吸管从塞子延伸到 t 形交叉软管连接器。</li>
	<li>使用乙烯基油管, 连接两个器 (图 1, #2 & #3), 并将第二个器的输出连接到 t 形交叉管接头。</li>
	<li>使用乙烯基油管, 连接第一个器的输入端口作为吸入端口 (图 1, #1)。</li>
	<li>使用乙烯基油管, 将傅书礼烧瓶的侧臂连接到气流调节器 (图 1, #7) 的输入端口和气流调节器的出口端到真空泵。</li>
	<li>根据图 2A中的示意图组装电路。</li>
	<li>通过串行适配器和制造商的软件, 将 PBASIC 程序诚 bs1 (图 2B, 也可在 https://github.com/ChastainAnderson/SVL) 上载到 BS1 微控制器 (图 1, #6)。</li>
	<li>将510螺纹底座 (图 1 #8) 置于环形支架夹具中。</li>
	<li>使用乙烯基油管, 将电磁阀 (图 1, #5) 连接到 t 型交叉管接头的自由端。 注: 该设备应完整, 并准备运行, 检查所有接头, 以确保他们是空气紧张, 并应用软管钳和真空润滑脂的需要。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56709/56709fig2.jpg"> 图 2: 电气示意图和 PBASIC 代码.图 2A显示用于装配所需电路的电气示意图, 以激活正常打开的电磁阀和按钮激活电子烟的加热线圈 (通过510螺纹电子烟罐底座)。加热线圈的电气参数 (P: 功率;R: 电阻;和 I: 电流) 的预测, 并应经验验证用万用表后组装。图 2B显示了在图 2A中控制电路所需的 PBASIC 计时程序 (也可在 https://github.com/ChastainAnderson/SVL 中使用)。定时常数 SVT & 间隙 (#5 & #6) 是以 ms 为单位, 并设置为2秒的激活时间, 并在二十八年代停机.<a href="//cloudflare.jove.com/files/ftp_upload/56709/56709fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 样品贮存和制备
<ol><li>在室温下, 将所有未开封的传统和电子卷烟样品存放在密闭的塑料袋中。</li>
	<li>一旦打开, 在 4 c 的密闭塑料袋中储存样品, 用纸巾吸收多余的水分。</li>
	<li>预平衡所有样品在室温下盒60% 湿度至少30分钟前使用。</li>
</ol>3. 卷烟烟雾/电子烟气溶胶萃取装置的一般操作
<ol><li>使用分析天平确定每个电子烟 cartomizer/罐前 vaping 的质量。前/后 vaping 重量的差异将用于确定适当的剂量。 注: 3R4F 参考卷烟被假定含有0.7 毫克的尼古丁, 而商业卷烟品牌的尼古丁含量可以用传统的分析方法13来确定。</li>
	<li>对于样品应用 1, 用4.3 毫升内皮细胞培养基填充原器的储层。样品应用 2, 用5毫升丙酮填充油层。</li>
	<li>
准备用于提取的电子香烟或常规香烟:
	<ol>
<li>如果使用传统的卷烟, 在过滤器周围应用一条清晰的胶带, 并在卷烟纸加入过滤器的地方放置一个容易看到的标记。</li>
		<li>如果使用香烟, 如电子烟, 确保电池是很好的充电和 cartomizer 紧拧到电池。</li>
		<li>如果使用电子香烟罐, 确保在油箱内装上适当数量的电子香烟液体, 并将油箱拧到510螺纹底座上。</li>
	</ol>
</li>
	<li>将常规或电子卷烟的尖端插入吸入式端口 (图 1, #1), 并用软管钳固定。</li>
	<li>打开真空泵。</li>
	<li>调整流量计, 以拉动1.65 升/分钟, 以确保55毫升粉扑超过2秒。</li>
	<li>打开微控制器。如果使用传统的香烟, 点燃香烟的第一个粉扑。</li>
	<li>运行, 直到预计的预期浓度 (在部分每百万或% 重量/体积) 达到。</li>
	<li>
使用分析天平确定每个电子烟 cartomizer/罐在汽化后的质量。将此测量与步骤3.1 中所采取的测量值进行比较, 以确定所消耗的总质量。计算溶剂的消耗质量/体积的浓度。使用所消耗的尼古丁的摩尔浓度, 使产品正常化。
	<ol>
<li>如果消耗的质量不足, 则将电子卷烟退回设备并进一步消耗。</li>
		<li>如果消耗了足够或过剩的质量, 请继续。</li>
	</ol>
</li>
</ol>4. 过滤和贮存
<ol><li>如果提取物是用于细胞培养, 过滤通过0.22 µm PES 注射器过滤器。</li>
	<li>立即使用提取物或存储在-80 c。作为准备安德森的一部分, et al.8, 电子烟气雾剂被证明是稳定的至少两个星期, 和两年的香烟烟雾的稳定性已建立由骗子, et al.13。</li>
</ol>5. 清洗设备
<ol><li>每次提取后, 用70% 乙醇和去离子水冲洗该装置的油管和储油层, 以防止样品之间的携带。</li>
	<li>在漂洗之后, 简要地运行空的设备允许气流帮助烘干线。</li>
</ol>6. 样品应用 1: 中性红吸收细胞活力测定
<ol><li>以4.3 毫升的内皮细胞生长培养基进行提取。</li>
	<li>一天前, 种子人脐静脉内皮细胞进入96井板, 密度为 1 x 104细胞/在100µL 内皮细胞生长培养基。</li>
	<li>用100µL 的新鲜内皮细胞培养基取代原有的内皮细胞培养基来治疗细胞, 以作为对照或75µL 的内皮细胞生长培养基, 混合25µL 2 毫米消耗的尼古丁浓度提取物 (1.4 毫克将尼古丁消耗到4.3 毫升内皮细胞生长培养基中), 最终浓度为500µM, 作为治疗。</li>
	<li>由于香烟烟雾和电子烟气溶胶中的许多成分都是挥发性的, 所以使用铝箔密封来保持油井的密闭。</li>
	<li>孵育板 24个 h 在37° c 和 5% CO2。</li>
	<li>
准备中性红染色溶液:
	<ol>
<li>创建一个100x 中性红股票解决方案, 溶解33毫克的中性红色染料到10毫升的缓冲盐溶液。</li>
		<li>使用前不久, 稀释100x 库存溶液1:100 在细胞培养基中创建1x 中性红染色液。</li>
		<li>孵育中性红染色液在37° c 至少30分钟之前使用和立即使用。 注意: 有些晶体在孵化过程中沉淀是正常的。应注意避免将这些晶体应用于细胞培养井。如果需要, 22 µm 过滤器可用于过滤中性红色股票和染色解决方案。</li>
	</ol>
</li>
	<li>去除提取物, 并添加100µL 中性红染色溶液每井, 使用过量创造至少三空白井为适当的量化。</li>
	<li>在37° c 和 5% CO2的 2-4 h 处孵育盘。</li>
	<li>去除中性红染色液, 在 PBS 中浸泡3x 冲洗。</li>
	<li>应用中性红脱污液 (50% 去离子水, 49% 乙醇, 1% 乙酸)。</li>
	<li>室温下至少孵育10分钟。</li>
	<li>读取 540 nm 的吸光度。</li>
	<li>通过将空白井的平均值减去平均值和正则化到空白调整控制井值的平均值来分析数据。</li>
</ol>7. 样品应用 2: 气相色谱质谱
<ol><li>在5毫升丙酮中进行提取。</li>
	<li>运行的设备, 以达到最终的浓度为100分之每百万 (重量的 e 液体消耗/丙酮体积) 的样品。</li>
	<li>使用精密玻璃注射器, 注入1µL 到注射器的 GC-MS 装置。在250° c 的条件下注入1:20 分裂比例的气相色谱/四极谱仪系统, 配有带有以下烤箱协议的 ZB-5 柱: 1 分钟在50° c;斜坡10° c/分至140° c;舷梯20° c/分钟到300° c 并且举行10分钟。</li>
	<li>将产生的质量谱与目标库相匹配, 以确定气溶胶成分。</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Reactivity+and+Respiratory+Toxicity+of+the+-Diketone+Flavoring+Agents:+2,3-Butanedione,+2,3-Pentanedione,+and+2,3-Hexanedione.">Chemical Reactivity and Respiratory Toxicity of the -Diketone Flavoring Agents: 2,3-Butanedione, 2,3-Pentanedione, and 2,3-Hexanedione.</a> Toxicol. Pathol. 44 (5), 763-783 (2016).</li><li>Cooperation Centre for Scientific Research Relative to Tobacco. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CRM No. 84 - Determination of Glycerin, Propylene Glycol, Water, and Nicotine in the Aerosol of E-Cigarettes by Gas Chromatographic Analysis. , (2017).</li></ol>

由杜兰大学管理的烟草产品管理科学研究金项目由奥客户服务管理事务处提供资金。

本协议最关键的元素是确保设备在每次提取的开始和结束时都是干净的, 并确保所有的密封都保持稳定, 以使气流保持一致。如果设备没有被正确地清洗, 有风险在样品之间运载。此外, 如果设备在长时间内不洁, 凝结气溶胶和干溶剂就会堵塞系统。请注意, 这是正常的, 有一个压力下降时, 膨化一个传统的卷烟和气流表应调整, 以提供所需的气流期间的粉扑, 而不是当设备是拉动房间空气。该方法的?...

尽管保健组织作出了集中的努力, 但烟草产品的使用仍然是全世界可预防死亡的主要原因, 其中大多数死亡归因于吸烟的1。自2003年进入市场以来, 电子卷烟在烟草产品用户中的知名度越来越高。目前, 电子香烟是最流行的替代传统香烟在美国成年人 (〜 5%)2和最流行的尼古丁传递系统中间 (〜 5.3%) 和高中生 (〜 16%)3。如果目前的趋势继续下去, 电子卷烟有望取代传统的香烟, 为子孙后代。然而, 电子卷烟使用的健康后果仍不清楚。
电子卷烟的研究直到 2013年3,4的电子卷烟普及程度迅速增加, 才开始认真。自那时以来, 采用了一些不同的模式来解决其毒性问题。然而, 许多研究的结果是相互矛盾的, 虽然电子卷烟的毒性一般低于传统卷烟, 但目前没有就电子香烟的健康后果达成共识使用5,6,7. 我们以前的研究表明, 电子卷烟对血管内皮的毒性要比传统的香烟低得多, 尽管它们有能力引起 DNA 损伤和诱导氧化应激和细胞死亡,8.然而, 在我们得出关于电子卷烟使用的健康后果的确切结论之前, 需要进行更多的研究。
由于常规卷烟是可预防的血管疾病的主要病因9, 因此对电子卷烟使用的血管健康风险的兴趣与日俱增, 如10、11、12。为了研究电子香烟对血管系统的影响, 我们的实验室开发了一个单片机操作的冒烟/vaping 设备 (图 1)8。该装置能够在水或有机溶剂中产生传统卷烟烟雾或电子香烟气溶胶的液体萃取物。由于气流由可调式气流调节器和 PBASIC 定时程序的组合控制, 因此该装置可根据任意数量的用户定义的协议来生成提取物。在这里, 我们详细介绍了该设备的组装和操作以及两个潜在的应用:体外细胞活力评估和气相色谱质谱。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56709/56709fig1.jpg"> 图 1: 吸烟/Vaping 设备.为吸烟/vaping 装置的物理装配示意图在香烟或香烟象电子香烟 (电子烟) 配置 (A) 和坦克电子香烟配置 (B)。组件键: 1) 吸入口;2) 初级收藏器;3) 溢流器;4) 傅书礼瓶真空疏水阀;5) 一般开式电磁阀;6) BS1 单片机;7) 气流调节器;8) 510 螺纹电子烟罐底座。<a href="//cloudflare.jove.com/files/ftp_upload/56709/56709fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>12 V AC/DC Wall Mount Adaptor</td>
			<td>Digi-Key</td>
			<td>T1099-P5P-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>2.2 Ohm Resistors</td>
			<td>Digi-Key</td>
			<td>A105635-ND</td>
			<td>Used in tandem to generate the 4.4 Ohm resistance in Figure 2A</td>
		</tr>
		<tr>
			<td>330 Ohm Resistors</td>
			<td>Digi-Key</td>
			<td>330QBK-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>510 Threaded Base</td>
			<td>NJoy</td>
			<td>N/A</td>
			<td>Recovered by dismantalling a second generation NJoy electronic cigarette</td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>Sigma-Aldritch</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone (Chromatography Grade)</td>
			<td>Sigma-Aldritch</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Stamp Project Board</td>
			<td>Digi-Key</td>
			<td>27112-ND</td>
			<td>This board contains the BS1 Microcontroller, serial adaptor, power switch, and a barrel pin connector for the AC/DC Wall Mount Adaptor</td>
		</tr>
		<tr>
			<td>Basic Stamp USB to Serial Adapter</td>
			<td>Digi-Key</td>
			<td>28030-ND</td>
			<td>An optional component to allow the BS1 serial adaptor to communicate through USB</td>
		</tr>
		<tr>
			<td>Buchner Flask (Vacuum Flask) 250 mL</td>
			<td>VWR</td>
			<td>10545-854</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Tape</td>
			<td>3M</td>
			<td>S-9783</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Vinyl Tubing, 3/8&#34; ID</td>
			<td>Watts</td>
			<td>443064</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Endothelial Cell Culture Medium</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-Aaper</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Regulator</td>
			<td>Dwyer</td>
			<td>VFA-23-BV</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Chromatograph</td>
			<td>Varian</td>
			<td>450-GC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Syringe, 10 mL</td>
			<td>Sigma-Aldritch</td>
			<td>Z314552</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Syringe, 10 &#181;L</td>
			<td>Hamilton</td>
			<td>80300</td>
			<td></td>
		</tr>
		<tr>
			<td>High Vacuum Silicon Grease</td>
			<td>Dow Corning</td>
			<td>146355D</td>
			<td></td>
		</tr>
		<tr>
			<td>Hose Clamp</td>
			<td>Precision Brand</td>
			<td>35125</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells</td>
			<td>ATCC</td>
			<td>PCS-100-013&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectrometer</td>
			<td>Varian</td>
			<td>300-MS</td>
			<td></td>
		</tr>
		<tr>
			<td>Midget Impinger</td>
			<td>Chemglass</td>
			<td>CG-1820-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red</td>
			<td>Sigma-Aldritch</td>
			<td>N4638</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Film</td>
			<td>3M</td>
			<td>PM-992</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Seal Roller</td>
			<td>BioRad</td>
			<td>MSR0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Seal; Foil</td>
			<td>Thermo</td>
			<td>276014</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Stand 20&#34;</td>
			<td>American Educational Products</td>
			<td>7-G15-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Solenoid Valve (normally open)</td>
			<td>US Solid</td>
			<td>USS2-00081</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid State Relay</td>
			<td>Digi-Key</td>
			<td>CLA279-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Stand Clamp</td>
			<td>Eisco</td>
			<td>CH0688</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, PES, 0.22 um</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>BD Syringe</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Through Hole Stopper, Size 6</td>
			<td>VWR</td>
			<td>59581-287</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>KNF Neuberger</td>
			<td>N86KTP</td>
			<td></td>
		</tr>
	</tbody>
</table>

a microcontroller operated device for the generation of liquid extracts from conventional cigarette smoke and electronic cigarette aerosol

电子卷烟是中、高中学生最受欢迎的烟草产品, 是成人中最受欢迎的替代烟草产品。对电子卷烟使用的后果进行高质量、重复性的研究, 对于了解新出现的公共卫生问题和制定基于证据的监管政策至关重要。虽然越来越多的论文讨论电子卷烟, 但在各组的方法上几乎没有一致性, 对结果的共识也很少。在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。本协议详细说明了该设备的组装和操作, 并演示了在两个示例应用程序中使用生成的提取物: 一个体外细胞活力测定和气相色谱质谱。该方法提供了一种直接比较传统卷烟和电子卷烟的工具, 是电子卷烟研究中一个容易进入的切入点。

在24小时内, 人类脐静脉内皮细胞暴露于传统的卷烟烟雾提取物 (EAE) 或电子香烟气溶胶提取物 (的), 有一个显着的 (控制与...... 控件 vs. EAE p< 0.01;n = 6) 细胞存活率降低 (图 3A)。提取物产生的膨化轮廓为 2, 2 秒, 55 毫升喷泡每分钟和正常化的基础上的摩尔浓度尼古丁的设备。暴露于500µM 消耗的尼古丁当量的学会极大地减少了活细胞到11.06 ±...

Watch this Scientific Journal Video about A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol at JoVE.com

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

在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。这种方法为直接比较传统卷烟和电子卷烟提供了一个有用的工具, 是电子卷烟研究中一个容易进入的切入点。

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 ...

a-microcontroller-operated-device-for-generation-liquid-extracts-from

Research

JoVE Journal

Bioengineering

8.3K 次观看.  Tulane University. 在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。这种方法为直接比较传统卷烟和电子卷烟提供了一个有用的工具, 是电子卷烟研究中一个容易进入的切入点。

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

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

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. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

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.  ...

科研

生物工程

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

生物电子界面衔接与Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

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 ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

关于纺织品有机电子设备的简单和可扩展的制作方法

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

从未引用的太平洋牡蛎生成虚拟序列的 cDNA 库的聚合策略

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...