The work was supported by EU through project FP7-PEOPLE-212-ITN 316832-OLIMPIA, 
Telethon - Italy (grants GGP12033 and GGP14022), Fondazione Cariplo (grant ID 2013-0738).

1.准备光活性基质<ol><li>准备一个P3HT:PCBM溶液（1:1重量/重量）的氯苯以20克/升的P3HT浓度。混合用磁力搅拌器将溶液至少4小时，在60℃。考虑150微升溶液的体积为每个衬底被制备。 </li><li>清洁的ITO涂覆的玻璃载片（R S = 10Ω/□，18×18毫米2，厚度170微米）的去离子水，丙酮和异丙醇中超声波仪连续浴（10分钟为每个工序）。干燥用氮气枪的盖玻片。把样品中的等离子体清洁器在100瓦的功率进行10分钟。 </li><li>沉积活性层的薄膜上经由旋转涂布清洁过的基材上。 <ol><li>用金刚石尖，切显微镜载玻片，（厚度≈1毫米），以在ITO涂覆的基材的相同的横向尺寸。卸下P3HT:从加热板PCBM溶液，让其冷却至环境温度。 </li><li>设置一个两圣EPS旋转涂布机程序:ⅰ）30秒以80​​0rpm; ⅱ）30秒1600转。放置在旋涂机的卡盘滑动，并开始真空进行修复。放丙酮在滑动下降，然后将清洗衬底，以在ITO涂覆侧朝上。启动旋转涂布机旋转甩掉多余的丙酮。 </li><li>把150微升的P3HT:PCBM溶液在基片上并再次开始旋转涂布机（使用在1.3.2中描述的相同的协议）。 </li><li>后旋涂机已完成，取出样品，小心地分离从载玻片上涂布的基材。清洁衬底的同一个洁净室拭子浸入丙酮背面​​。 注意:光活性基材可以直到用于存放几天在黑暗的框。对于更长的时间，让他们在与惰性气体的手套箱。 </li></ol></li></ol> 2.准备细胞培养基和电解决方案<ol><li>制备完全生长培养基（CGM），用于HEK-293细胞:杜尔贝科改良伊格尔培养基（DMEM），补充有10％胎牛血清（FBS）的未热灭活的，100U / ml青霉素和100μg/ ml链霉素。消毒用0.2微米的过滤系统和存储介质在4℃下。 </li><li>准备在超纯水（以mM浓度）的胞外溶液:氯化钠135，氯化钾5.4，5 HEPES，10葡萄糖，1.8 氯化钙 2，1 MgCl 2的。调节pH至7.4，用NaOH。消毒在4℃的溶液用0.2微米的过滤系统和存储。 </li><li>准备在超纯水中的细胞内溶液（浓度以mM计）:12的KCl，125 K-葡萄糖，1 MgCl 2的，0.1 氯化钙 2，10 EGTA，10 HEPES，10的ATP 的 Na 2。需要注意的是三磷酸腺苷的 Na 2是热敏感和最后添加。调节pH至7.4，用NaOH。消毒在-20℃在1毫升管中的溶液用0.2微米的过滤系统，等分和存储。 </li></ol> 3.电镀的HEK-293细胞的在活性基底<ol><li>放置在覆盖有铝或封闭的陪替氏培养皿，并把在烘箱中于120℃进行2小时的灭菌玻璃烧杯光敏衬底。从这一刻起，在无菌条件下进行的所有操作。把样品在无菌罩，让他们冷静下来。 </li><li>涂层光活性基片的粘合层，以减少表面疏水性，并促进细胞粘附。 <ol><li>放置在一个P35培养皿的活性基片，或将6多孔板。给出的共轭聚合物层的疏水性，使聚二甲基硅氧烷（PDMS）以及固定样品和限制的解决方案。 <ol><li>混合PDMS和催化剂在10:1的比例用玻璃棒，得到粘稠浆液。 </li><li>倒在一个6多孔板浆料，分配的量足以覆盖一半的每个孔。 </li><li>等待PDMS聚合反应在室温。 </li><li>切固化硅橡胶，中间为obta寄存器中聚合物样品的相同尺寸的正方形。 </li><li>为至少30分钟灭菌的PDMS孔浸在70％的乙醇 - 水混合物中。 </li></ol></li><li>如果使用的是PDMS好，刮沿着井用尖头镊子的整个边界的光活性层，为了做好后细胞培养取出PDMS时避免整个层的分离。 </li><li>覆盖纤连蛋白溶液（2毫克/升的PBS）各样品的整个表面上。每个样品500-750微升之间的体积通常是足够的，如果一个PDMS以及使用（井明面上:15×15 平方毫米）。如果没有PDMS好，约2毫升纤维连接蛋白的解决方案是必要的，完全沉浸样品;注意，虽然在溶液的基材不浮动。孵育样品在37℃下至少1小时。 </li><li>取出用玻璃吸管纤连蛋白溶液，并用2毫升的PBS洗涤一次。 </li></ol></li><li> PL吃HEK-293细胞上的纤连蛋白处理的样品以15,000细胞/ cm 2，在完全生长介质的密度。使用750-1,000微升CGM的体积为每个样品如果一个PDMS井的情况下，否则CGM 2-3毫升是必要的。孵育细胞，在37℃和5％CO 2的24-48小时。 </li></ol> 4.光刺激方案和电生理学测量<ol><li>放于水浴中的胞外溶液，在37℃至热能化;解冻细胞内的解决方案，把它放在1毫升注射器用34号针头，并保持与冰的接触，以避免ATP的降解。 </li><li>采取从孵化器细胞的基板，仔细去除PDMS以及如果存在的话。取出用玻璃吸管生长培养基和冲洗用胞外溶液的样品。缓慢进行，以避免细胞脱落。 </li><li>把设备插入到电车站与细胞外搜索解决方案的样品架n和参考电极。准备新鲜的玻璃微的膜片钳与拉马（理想的电阻范围为2-4MΩ）。填写一半的吸管与细胞内的解决方案，并将其安装在显微操作。 </li><li>寻找一个健康细胞来修补设备上。为了避免光活性基片的不需要的激励，则使用红外线照明进行成像，将长波长通滤波器（例如，一个切波长λ= 750纳米，可用于聚合物吸收在可见）中的显微镜照明器的前方。 </li><li>修补所选择的小区和通过经由显微镜（图1）的荧光激发路径传送光到样品申请光学刺激所需的协议。膜片钳协议的详细描述可以在参考20中找到。 <ol><li>膜片吸管靠近定位于细胞膜用测微操纵器。在定位，适用overpressur以e为吸移管，以避免在刀尖上的污垢粘附。移液器应该是几微米的单元格上方。 </li><li>开始降低吸移管向细胞膜，同时控制上的补丁控制软件移液器阻力。当吸液管电阻已增加大约1兆欧，从吸管同时施加轻柔的吸力，以形成所述吸液管和细胞膜之间的千兆欧密封移除超压（即，所测量的电阻应增加在大于1的值GΩ） 。 </li><li>施加负电位加到吸管接近预期细胞静息电位（对HEK293细胞的-40电位毫伏可以使用），以帮助稳定密封。补偿电容瞬变由于吸管电容与贴片上的放大器和/或补丁控制软件的相对的命令。 </li><li>打破隔膜，使电进入细胞质通过施加短暂而强烈的吸力脉冲到吸管。设置补丁放大器电流钳模式（I = 0，即，没有电流注入细胞）来跟踪细胞膜电位。等待几分钟，看细胞电位是稳定的。 </li><li>应用所需的照明协议向小区和记录在膜电位的影响。 注意:照明可以被传递到样品或者从底部或从顶部，视显微镜架构。 <ol><li>选择的激发波长是深受活性材料吸收;对于P3HT:PCBM共混，在450-600纳米范围内的波长都可以使用。合适的光激发密度的范围是10-100毫瓦/毫米2。 注意:光的短脉冲（以下:10-20毫秒）结果在小区的一过去极化，而与长期照明（几百毫秒或更长的脉冲）的持续超极化观察直到灯被关闭。 </li></ol></li&gt; </ol></li></ol>

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The authors have nothing to disclose.

关键步骤所报告的协议为体外细胞光学刺激主要涉及光敏聚合物的选择，热灭菌参数，强度和光刺激的持续时间。一个P3HT:PCBM薄膜在这里被选中，因为它保证了良好的时间和电化学稳定性。然而，人们应该注意到，并非所有的感光聚合物可提供模拟表演，22更具体在照明。此外，在这种情况下，选定的加热杀菌参数不导致的聚合物光电特性相当大的退化;通知然而，在情况下，灭菌?...

能够操作与精确的空间和时间分辨率的细胞活性的可能性表示在神经科学研究和在神经和精神障碍的治疗的关键策略1的传统方法是基于使用电极细胞电刺激定位成接近或接触目标系统，2可以是不同的复杂性（单电池，蜂窝网络，脑切片， 在体内脑组织）。在过去的一个世纪中，利用膜片钳，金属基板集成电极所提供的生理学和神经网络的运作机制的单个神经元和病理生理学的详细图片。然而，电刺激患有重要的限制。第一个涉及一种普遍较差空间分辨率由于电极和它们的固定几何形状的的物理尺寸，这是不能容易地适应像生物组织复杂的组织系统。另外，有关的刺激和记录系统之间的串扰的电极阻抗和问题可能恶化的测量值的最终信噪比3另一方面，利用光的刺激可能有助于克服许多局限性电方式。首先，它提供了前所未有的空间（&lt;1微米）和时间分辨率（&lt;1毫秒），使得能够针对特定的细胞类型或甚至亚细胞区室。此外，它是高度非侵入性，因为它避免了与感兴趣的组织的任何物理接触和disentangles刺激从记录。而且，无论光的强度和波长，可以精确地调节，从而多样化刺激方案都可以应用。3,4- 然而，动物细胞中的绝大多数不呈现给光任何特定的灵敏度。几种策略用于光学stimulation的因而被提出，或者利用附近或在细胞内的感光分子介质，或使用光敏器件外部设置的，靠近小区。前一类是指内生机制，例如通过可见光或红外光（IR）的刺激，以及利用或者光异构化/光解的化合物或光敏分子致动器（光遗传学）基因的表达。后一类包括技术实现了与使用无机纳米/微米颗粒或光电的硅衬底的外源性刺激。5然而，所有这些系统都具有光明的两侧和缺点。具体地，细胞在可见光范围内源性的吸收弱，不可靠，以及伴随的产生活性氧的物质可以是有害的细胞。在一般情况下，红外用于诱导局部热加热因吸水，但水的消光系数小，因此要求圣荣红外光（从几十到几百瓦/毫米2），它是难以通过标准显微镜光学器件以提供并可能造成安全问题为在体内的应用程序。另一方面，光笼蔽切换化合物具有时间限制的动作，往往需要UV光，是很难传递由于有限的组织穿透。此外，他们还从激活的化合物后，光解照射范围外扩散问题的困扰。最后，光遗传学工具使得科学家能够针对特定的细胞亚群和副舱室，并迅速成为的关键技术在神经科学研究之一。然而，通过病毒载体中插入的外源DNA区段提出了重要的安全问题，特别是考虑通过对人类患者。5,6-出于这些原因，新材料和设备能够细胞的光学操纵研究是一个非常热门的话题。 最近，一种新型基于使用的光敏共轭聚合物，能够有效地转导的光刺激成细胞电活动的调制手段，已经提出了。由聚合物光激发的细胞刺激（CSPP）技术利用许多键支持特性的典型的有机半导体:它们在本质上是敏感的光在可见光范围内; 7它们是生物相容的，柔软和适形和它们的机械灵活性允许与组织亲密的接口无论是在体外 和体内8-10。除此之外，他们可以很容易地官能化，以更好地适应与活细胞的界面，并启用特定激励，探测和感测能力。11,12而且，它们支持电子以及离子迁移，从而使它们适合的组合电子广告生物学13,14有趣的是，他们可以在光伏模式下工作，避免了需要应用外部偏置˚F或有效的细胞光刺激。15 CSPP技术的可靠性已在几个系统先前已经证实，包括原代神经元，星形胶质细胞15,16，17二次电池线 18和取出的视网膜组织16在这项工作中，所有必要的步骤，以制造光敏感的生物聚合物的接口 19 在体外系统的光学刺激进行详细说明。作为研究的情况下，区域规则性聚（3-己基噻吩）的一个典型的有机光生伏打共混物（RR-P3HT），用作电子给体，和苯基C61-丁酸 - 甲基酯（PCBM），充当电子受体采用。作为生物系统，人胚肾（HEK-293）细胞中被使用。设置有细胞的活性通过电生理学测量的相对记录一个光刺激协议的一个例子。 所描述的平台然而一般有效性，并且它可以很容易地扩展到使用其它共轭聚合物（通过适当地调节溶液的制备方法和淀积参数），不同类型的细胞（通过适当改变细胞培养协议，电镀过程和时间请求细胞接种和增殖）和不同的刺激方案（光的波长，刺激频率和持续时间，光激发密度）。

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rr-P3HT</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">698989-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ITO-coated substrates</td>
			<td style="width:120px;">Nano-CS</td>
			<td style="width:191px;">IT10300100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chlorobenzene</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">319996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCBM</td>
			<td style="width:120px;">Nano-C</td>
			<td style="width:191px;">Nano-CPCBM-BF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acetone&#160;</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">270725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isopropyl alcohol</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">563935</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEK cells</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">P5244</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-MEM</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-30-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">E8008-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FBS</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-30-2020</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical control of living cells electrical activity by conjugated polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

细胞可以在P3HT容易培养:PCBM基板，提供一个合适的粘附层沉积（如在所描述的协议的步骤3.2中使用的纤连蛋白）。 P3HT:PCBM光学吸收峰在可见光谱的绿色部分;然而其他光敏感共轭聚合物可以被选择，根据优选光刺激的波长范围（ 图2）。这些基板的生物相容性已经证明不仅与细胞系18,21一样的HEK-293，也可与神经元15和星形胶质细胞的原代培养<sup...

Watch this Scientific Journal Video about Optical Control of Living Cells Electrical Activity by Conjugated Polymers at JoVE.com

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

由共轭聚合物活细胞电活动的光控制

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

optical-control-living-cells-electrical-activity-conjugated

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Istituto Italiano di Tecnologia. A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

视频: Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

科研

生物工程

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

通过光纤使用波前整形多个信号的传输

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

用超常光透射法定量测定人血清中的心脏标志物

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

Nanoplasmonic 光学晶格中微粒子的俘获

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.