This research was supported in part by a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences.

1.实验装置和样品制备<ol><li>使用光刻，蚀刻和如先前6所述熔化过程制造microtoroids。制造上的硅晶片（芯片）的顶部microtoroids通常具有80-100微米的大直径，以及2微米的小直径。 </li><li>放松大致从它的纤维卷轴的米单模光纤（125微米包层，4.3微米的模场直径）。 </li><li>在光纤的开卷部分的中间，剥去光纤使用剥线围绕聚合物涂层的一小部分（2.5厘米）。注意:这是光纤，其将被用来渐逝地将光耦合到所述微型环的部分。 </li><li>清洁剥离的纤维用异丙醇酒精和无尘擦拭。 </li><li>持使用由磁性压块的一个光纤夹持该部分的纤维就位。 </li><li>薄的剥离纤维〜500nm下meltin克，并用氢气火炬和两个步进电机在相反的方向移动，在60微米/分钟牵引。光纤定位火焰的顶端部，它应该是约10毫米高的内部。停止拉光纤时通过光纤传输将停止脉动，它可以通过视觉监视（通过观察即从纤维眨眼侧向散射和关闭光），或通过光纤连接到其连接到示波器的光电二极管。 </li><li>切割光纤的一端，并把它插入到裸光纤适配器。放置光纤的这一端进入光接收器的输入端。 </li><li>纤维耦合光纤线轴到激光的通过使用一个光纤耦合器的另一端。 </li><li>放置使用任一环氧树脂或双面胶带的微型环芯片上的样品架的顶部（不锈钢，37.8毫米×6.4毫米×3.2毫米）。 </li><li>装入样品支架上的定位台，其包括3轴纳米定位的顶部（正anocube）阶段（参照设备列表）上的3轴千分尺的顶部。执行一个气动隔离光表中的所有实验，以减少震动。 </li><li>粗略位置使用3轴千分尺的样品芯片。 </li><li>对准芯片平行于光纤使用纳米定位器的含微型环-。注意:输入光（〜633纳米）中的一个波长的距离内对齐微型环。为这一过程的可视化使用两个成像列（管与物镜和相机，见设备清单），其定位在顶部和在芯片的一侧。 </li><li>优化通过光纤使用一种在线偏振控制器引导所述激光的偏振（见设备列表）与旋钮调节极化。注意:当在光纤的传输所测量的倾角出现最窄最佳偏振的实现。注意这浸在示波器（详情参见步骤2.2）。 </li><li>构造通过环氧树脂将玻璃盖玻片上使用玻璃显微镜载玻片作为间隔物样品台上的样品室。注:有机玻璃外壳覆盖了整个安装程序可能最大限度地减少气流非常有用。一个小的开放应该留给允许的解决方案中使用管道进行抽水。 </li><li>热平衡粒子混悬液或单分子水溶液为≥1小时在室温水浴（〜500毫升）。注:样品使用从制造商， 例如 ，PBS或HEPES中指定的相关联的缓冲器稀释至所需浓度在微量离心管中。如果没有检测到结合事件，增加缓冲液的盐浓度。 </li><li>含有溶液（1毫升）中简要地为〜2秒涡旋粒子。 </li><li>注入含粒子溶液到样本室以1ml / min，使用1ml注射器泵。 </li><li>后样品室填补，关闭注射器泵。 </li><li>等待30秒前记录数据，以减少从流体流动会影响测量所得的机械振动的影响。 </li></ol> 2.频率锁定<ol><li>重新耦合圆环到光纤通过移动样品支架与纳米定位，因为耦合将因受到干扰到注射液。 </li><li>由通过各种波长的扫描计算机控制的输入激光定位微型环的谐振波长。执行此步骤通过发送一个三角波电压信号，以调整所述激光的波长的激光控制器内的压电元件。执行使用可见光（635纳米±2.5纳米），因为是光在水在此波长吸收少的实验。 </li><li>通过插入的光纤的输出入一个自动均衡光器测量的光透射通过光纤。插入受光的输出转换成使用BNC电缆的示波器。观察Øn中的示波器，在该微型环的谐振波长，通过光纤传输下降。 </li><li>附加光接收器的输出对所述频率通过电缆锁定反馈控制器（见设备清单）的主输入端。 </li><li>采用顶级的高峰期为2 kHz的抖动频率和19个FM波长振荡的幅度锁定在运行模式下自动锁定的频率锁定反馈控制器。经验设定在使用齐格勒- Nichols整定规则14的软件窗口的比例-积分-导数设置。注意:这些值只需要在所有实验的开始设置一次。 </li><li>自动锁定激光器的波长到微型环的谐振波长。执行填充样品室之后此步骤。注意:如果波长偏移过大，则反馈控制器将失去锁定并自动切换到扫描模式以找到谐振波长位置。这OC小人的波长偏移超过约一线宽（至少600 FM这里考察的所有系统）放大。 </li><li>使用24位数据采集卡上记录所述反馈控制器的输出在20千赫。导出的数据，经由数据采集软件的文本文件。 </li></ol> 3.数据处理和分析<ol><li>傅立叶变换在MATLAB中的数据。 </li><li>低通使用"砖墙"滤波器的截止在1 kHz，除去2千赫强加抖动频率的数据（见补充代码文件截图1）。 </li><li>采用16赫兹的窗口大小计算陷波滤波器的数据。注意:这样做是为了除去已知噪声源，在这种情况下，60赫兹电子线路噪声和谐波，以及100赫兹（从激光驱动未来）和它的谐波（见补充编码文件截图1）。 </li><li>逆傅立叶变换的数据回时域。 </li><li>值滤波器使用窗口中的数据1,001样品尺寸（见补充代码文件截图2）。 </li><li>定位使用Kerssemakers 等人 15的步骤寻找算法步骤中的变化谐振波长。 </li><li>生成每个结合步骤的幅度的直方图。 </li><li>使用公式计算粒径。 （1）（见讨论）。 </li></ol>

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The authors declare that they have no competing financial interests.

作为粒子结合，的环形增大的谐振波长 （λ）。如果粒子解除绑定，谐振波长相应减少（降压事件）。粒径（d）可以通过各波长步骤的幅度的直方图来确定。每个波长台阶的高度而变化，由于结合的粒子的尺寸变化和由于对那里的粒子结合的微型环的位置。当颗粒粘合在微型环其中电场（E 0，max）为最高的赤道在共振波长（步骤高度）的最大变化发生。这个最大阶梯高度（Δλ）是相关的粒径通过公式（1）8， 其中 a是颗粒半径，D是根据绑定粒子的折射和其周围介质的折射率的介电常数，V m是二极管灯的模体积的微型环内吨通过有限元模拟2来确定， 以及 E 0（R s）是电场在粒子赤道通过有限元模拟还确定的幅度: <img alt="式（1）" src="/files/ftp_upload/53180/53180eq1.jpg" width="500" />反转式。 （1）表示的信号强度（Δλ）秤与颗粒体积（3）。我们的介电系数定义为: <img alt="公式（2）" src="/files/ftp_upload/53180/53180eq2.jpg" width="150" />其中是折射周围介质的折射率，并且是指数折射颗粒。基于式（1）以及额外的直方图和大小校准为粒径理论估计算结果列于2，16。 花可以通过增加在其频率锁定反馈控制器跟踪微型环的波长的频率进行修改快速跟踪。数据处理过程可以通过使用移动平均，而不是中值滤波器进行修改，结合事件仍可以恢复，但中值滤波器使台阶边缘是较好的保存。这种技术的局限性包括事实的微型环的颗粒时结合波长偏移取决于那里的谐振器的粒子土地。因此，确认的单个颗粒的结合的依赖于许多离散的结合事件的直方图的生成。如果没有检测到不同的结合事件，增加了溶剂的盐浓度有所帮助。 这种技术对于现有方法的意义在于，没有需要标签来询问目标分子。然而，选择性结合需要官能与抗体的传感器。其它的优点包括这样的事实，由于微型环谐振器具有更大的捕获区相比，高灵敏度的表面等离子体共振方法，粒子结合事件更可能发生。此外，由于花并不要求可光漂白荧光标签，花能够长（&gt; 10秒）测量具有快速（毫秒）的时间分辨率。 在协议中的关键步骤包括对准光纤锥与微型环。一旦环形浸渍于通过液体的纤维的液体，运动过多可能导致锥形断裂，从而结束了实验。花目前的措词，因此不适合在小时的时间规模实验。此外，一旦微型环一直沉浸在液体和颗粒结合，所述品质因数（Q）无可挽回滴过的小时和峰值升时间尺度ocking最终可能会变得不稳定。在这种情况下，一个新的设备是必需的。因为我们抖动我们的激光频率在一个很小的范围内围绕共振峰，花不同时在整个共振谱扫描，因此不测量实时质量变化因素，因为颗粒结合。综观之前，只有少数颗粒的结合后的品质因数，我们没有看到显著Q因子的降解。我们预期这是因为原来的原始环形具有相对低的Q因数（载Q中的〜1×10 5 -5×10 6的水）。 我们注意到，激光诱导波动噪声中减去出使用自动平衡光器。我们通过将光纤直接接触的微型环尽量减少光纤对环形的波动。此外，如果PID参数设置不正确，波动就会出现，也就是说，系统将不会快速accurat伊利轨道波长偏移。齐格勒-尼科尔斯调整规则，可以用来设置正确的PID设定14。按照此处列出的步骤，它应该是能够检测和尺寸的纳米粒子，从几百纳米至几纳米，包括单一的生物分子。

单分子检测的实验是用于降低用于疾病的早期检测的分析物的生物传感器的使用量，是有用的，并且用于检查分子3的基本性质。这种实验是使用标签，但是，标签不总是能够获得对特定蛋白质，增加了成本，可能扰乱正在研究中的事件，并且可以是不方便，尤其是即时现场实验或点OF-典型地护理诊断。 目前金标准的无标记生物传感器是表面等离子体共振4，但是商业表面等离子体共振系统通常具有检测纳米的量级的典型下限。近日，光学谐振器已成为一个有前途的技术，无标签的单分子生物检测5。基于长期（NS）光学谐振器的工作光6,7限制。光是渐逝典型地通过光纤耦合到这些设备。当经过光纤的光的波长的谐振器的谐振波长相匹配时，点亮有效地耦合到谐振器。此耦合光谐振器的空腔中的谐振器的周附近产生的渐逝场中的全内反射。作为颗粒进入渐逝场和绑定到谐振器中，谐振器的变化成比例地粒子8的容积的谐振波长。 在检测能力方面，微谐振器前面已用于检测单个A型流感病毒颗粒（100纳米）9,10。最近，plasmonically增强微球的光学谐振器已经被用于检测的单牛血清白蛋白分子 11和8-mer的寡核苷酸12，但是这种方法限制了粒子捕捉区域至0.3μm2每日副。面积较大的捕捉生物传感器是理想的最大化粒子探测的机会。当前的解决方案为基础的无标记生物传感技术与大（&gt; 100微米2）捕获的区域被限制于检测到聚苯乙烯颗粒≥25纳米。 我们已开发了一种基于光谐振器技术被称为频率锁定光学耳语倏逝谐振器（花）13（图1）一个无标记生物传感系统，其能够时间分辨单分子检测在溶液中的。花使用微型环光谐振器结合频率锁定反馈控制，平衡检测，并计算滤波以向下检测小颗粒以单个蛋白质分子的长光子寿命。使用频率锁定的允许系统始终跟踪微型环的移位共振作为颗粒结合，而无需扫或扫描激光波长过大范围。可以使用花的原理来增强的其他技术的检测能力包括电浆增强。在下文中，该程序用于执行FLOWER进行说明。

<table><tbody><tr><td>Tunable diode laser</td><td>Newport</td><td>TLB-6300</td><td></td></tr><tr><td>Laser controller</td><td>Newport</td><td>TLB-6300-LN</td><td></td></tr><tr><td>Frequency locking feedback controller</td><td>Toptica Photonics</td><td>Digilock 110</td><td></td></tr><tr><td>Auto-balanced photoreceiver</td><td>Newport</td><td>Model 2007</td><td></td></tr><tr><td>In-line polarization controller</td><td>General Photonics</td><td>PLC-003-S-90</td><td></td></tr><tr><td>24-bit data acquisition card</td><td>National Instruments</td><td>NI-PCI-4461</td><td></td></tr><tr><td>Recombinant human interleukin-2</td><td>Pierce Biotechnology</td><td>R201520</td><td></td></tr><tr><td>20 nm polystyrene beads</td><td>Thermo Scientific</td><td>3020A</td><td></td></tr><tr><td>NanoCube XYZ Piezo Stage</td><td>Physik Instrumente</td><td>P-611.3</td><td></td></tr><tr><td>Optical table</td><td>Newport</td><td>VH3660W-OPT</td><td></td></tr><tr><td>Objective lens for imaging column</td><td>Navitar Machine Vision</td><td>1-60228</td><td></td></tr><tr><td>Imaging column (adaptor tube)</td><td>Navitar Machine Vision</td><td>1-60228</td><td></td></tr><tr><td>High-Res CCD camera for imaging column</td><td>Edmund Industrial Optics</td><td>NT39244</td><td></td></tr></tbody></table>

label-free single molecule detection using microtoroid optical resonators

检测低浓度的分子到单分子限制对如能早期发现疾病，并在分子的行为基础研究领域的影响。单分子检测技术通常利用诸如荧光标签或量子点，标签然而，标签不总是可用的，增加了成本和复杂性，并可能扰乱所研究的事件。光学谐振器已成为一个有前途的手段来检测单分子，而不使用标签。目前在溶液通过非plasmonically增强裸光谐振器系统检测到的最小的颗粒是25纳米的聚苯乙烯球1。我们已经开发了一种称为频率锁定光学花落逝谐振器（花），可以在水溶液中2超过这个限制，实现无标记单分子检测的技术。由于信号强度与尺度颗粒体积，我们的工作表示&gt; 100X improveme核苷酸中的信号，以对现有技术的当前状态的噪声比（SNR）。这里后面FLOWER的程序都在努力提高在该领域的使用情况。

粒子结合事件清楚地视为阶梯状变化微型环的谐振波长随时间（图2A）。这些步骤的高度被示为在图2B中的直方图。 图2-4示出从外来体的结合代表迹线（纳米囊泡），5纳米二氧化硅珠，和单人白细胞介素2分子，分别。该阶梯状的事件比例与粒径的事实表明，该技术已被正确地执行。这可以通过产生的台阶高度（图2B）的直方图和比较观察到的理论预测的最大台阶高度进行分析，如下面讨论的。 <img alt="图1" src="/files/ftp_upload/53180/53180fig1.jpg" /> 环形感应系统的 图1. 框图。从调谐二极管激光光被分W¯¯<html>第i通过光纤耦合光进入环形，并直接发送到一个自动均衡光器的一个输入端的另一部分中发送的部分。该光纤的输出被发送到自动平衡光接收器的第二输入。光接收器的输出被发送到调制该激光定位微型环的共振波长的值的反馈控制器。作为粒子结合于环形，共振频率偏移。激光的波长和微型环的谐振波长之间的差被发送到比例-积分-微分控制器，其允许激光尽可能快圆环的波长匹配和尽可能顺利。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53180/53180fig1large.jpg" target="_blank">请按此查看大版本这个数字。</a> <html> p_upload / 53180 / 53180fig2.jpg"/&gt; 随着时间的推移20纳米珠子结合到微型环的表面 图2. 共振波长的变化。（A）的移位的微型环的谐振波长随着时间的推移20纳米珠子结合到表面。高度的每个谐振波长一步事件（振幅）（二）直方图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53180/53180fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53180/53180fig3.jpg" /> 随着时间的推移作为单个外来体结合到微型环的表面上。图3的共振波长的变化。个别结合事件被视为随着时间的推移离散变化（步骤）中的谐振波长。"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53180/53180fig4.jpg" /> 随着时间的推移5nm的硅珠图4.共振波长变化结合到微型环的表面上。颗粒通过被动吸附粘附在环形表面。粒子结合事件被视为在环形随时间的谐振波长离散的步骤。粒子解吸被看作是一个向下的台阶。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53180/53180fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53180/53180fig5.jpg" /> 随着时间的推移，IL-2分子图5.共振波长变化结合到微型环的表面上。大分子结合事件被看作是在谐振波长随着时间的推移离散的步骤。这些步骤看起来类似于在图4中的两种类型的颗粒是大致相同的尺寸。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53180/53180fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

label-free-single-molecule-detection-using-microtoroid-optical

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.2K 次观看.  California Institute of Technology. We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented. 

视频: Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Fabrication of Silica Ultra High Quality Factor Microresonators

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the quality factor (Q) of the cavity, can be in excess of 500ns for cavities with Q factors above 100 million. As a result of their low material losses, silica microcavities have demonstrated some of the longest photon lifetimes to date1-2. Since a portion of the circulating light extends outside the resonator, these devices can also be used to probe the surroundings. This interaction has enabled numerous experiments in biology, such as single molecule biodetection and antibody-antigen kinetics, as well as discoveries in other fields, such as development of ultra-low-threshold microlasers, characterization of thin films, and cavity quantum electrodynamics studies.3-7
The two primary silica resonant cavity geometries are the microsphere and the microtoroid. Both devices rely on a carbon dioxide laser reflow step to achieve their ultra-high-Q factors (Q&gt;100 million).1-2,8-9 However, there are several notable differences between the two structures. Silica microspheres are free-standing, supported by a single optical fiber, whereas silica microtoroids can be fabricated on a silicon wafer in large arrays using a combination of lithography and etching steps. These differences influence which device is optimal for a given experiment.
Here, we present detailed fabrication protocols for both types of resonant cavities. While the fabrication of microsphere resonant cavities is fairly straightforward, the fabrication of microtoroid resonant cavities requires additional specialized equipment and facilities (cleanroom). Therefore, this additional requirement may also influence which device is selected for a given experiment.
Introduction
An optical resonator efficiently confines light at specific wavelengths, known as the resonant wavelengths of the device. 1-2 The common figure of merit for these optical resonators is the quality factor or Q. This term describes the photon lifetime (&tau;o) within the resonator, which is directly related to the resonator's optical losses. Therefore, an optical resonator with a high Q factor has low optical losses, long photon lifetimes, and very low photon decay rates (1/&tau;o). As a result of the long photon lifetimes, it is possible to build-up extremely large circulating optical field intensities in these devices. This very unique property has allowed these devices to be used as laser sources and integrated biosensors.10
A unique sub-class of resonators is the whispering gallery mode optical microcavity. In these devices, the light is confined in circular orbits at the periphery. Therefore, the field is not completely confined within the device, but evanesces into the environment. Whispering gallery mode optical cavities have demonstrated some of the highest quality factors of any optical resonant cavity to date.9,11 Therefore, these devices are used throughout science and engineering, including in fundamental physics studies and in telecommunications as well as in biodetection experiments. 3-7,12
Optical microcavities can be fabricated from a wide range of materials and in a wide variety of geometries. A few examples include silica and silicon microtoroids, silicon, silicon nitride, and silica microdisks, micropillars, and silica and polymer microrings.13-17 The range in quality factor (Q) varies as dramatically as the geometry. Although both geometry and high Q are important considerations in any field, in many applications, there is far greater leverage in boosting device performance through Q enhancement. Among the numerous options detailed previously, the silica microsphere and the silica microtoroid resonator have achieved some of the highest Q factors to date.1,9 Additionally, as a result of the extremely low optical loss of silica from the visible through the near-IR, both microspheres and microtoroids are able to maintain their Q factors over a wide range of testing wavelengths.18 Finally, because silica is inherently biocompatible, it is routinely used in biodetection experiments.
In addition to high material absorption, there are several other potential loss mechanisms, including surface roughness, radiation loss, and contamination loss.2 Through an optimization of the device size, it is possible to eliminate radiation losses, which arise from poor optical field confinement within the device. Similarly, by storing a device in an appropriately clean environment, contamination of the surface can be minimized. Therefore, in addition to material loss, surface scattering is the primary loss mechanism of concern.2,8
In silica devices, surface scattering is minimized by using a laser reflow technique, which melts the silica through surface tension induced reflow. While spherical optical resonators have been studied for many years, it is only with recent advances in fabrication technologies that researchers been able to fabricate high quality silica optical toroidal microresonators (Q&gt;100 million) on a silicon substrate, thus paving the way for integration with microfluidics.1
The present series of protocols details how to fabricate both silica microsphere and microtoroid resonant cavities. While silica microsphere resonant cavities are well-established, microtoroid resonant cavities were only recently invented.1 As many of the fundamental methods used to fabricate the microsphere are also used in the more complex microtoroid fabrication procedure, by including both in a single protocol it will enable researchers to more easily trouble-shoot their experiments.

We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids. The reflow method removes surface imperfections, allowing long photon lifetimes within both devices. The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to biodetection.

硅胶超高品质因子微谐振器的制造

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the ...

科研

工程学

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

纳米粒子的光学捕获

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

折光传感荧光芯微腔的合成和操作

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

Implementation of a Reference Interferometer for Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing its free spectral range (FSR), the module is put in parallel with a fiber taper-microcavity system and then calibrated through isolating and eliminating random shifts in the laser frequency (i.e. laser jitter noise). To realize the taper-microcavity junction and to maximize the optical power that is transferred to the resonator, a single-mode optical fiber waveguide is pulled. Solutions containing polystyrene nanobeads are then prepared and flown to the microcavity in order to demonstrate the system&#8217;s ability to sense binding to the surface of the microcavity. Data is post-processed via adaptive curve fitting, which allows for high-resolution measurements of the quality factor as well as the plotting of time-dependent parameters, such as resonant wavelength and split frequency shifts. By carefully inspecting steps in the time-domain response and shifting in the frequency-domain response, this instrument can quantify discrete binding events.

A reference interferometer technique, which is designed to remove undesirable laser jitter noise for nanodetection, is utilized for probing an ultra-high quality factor microcavity. Instructions for assembly, setup, and data acquisition are provided, alongside the measurement process for specifying the cavity quality factor.

一个参考干涉仪的Nanodetection实施

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing ...

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

制造和微流控光机振荡器测试

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

生物工程

Fabrication and Operation of a Nano-Optical Conveyor Belt

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological and physical sciences over the past few decades. The progress made in this field invites further study of even smaller systems and at a larger scale, with tools that could be distributed more easily and made more widely available. Unfortunately, the fundamental laws of diffraction limit the minimum size of the focal spot of a laser beam, which makes particles smaller than a half-wavelength in diameter hard to trap and generally prevents an operator from discriminating between particles which are closer together than one half-wavelength. This precludes the optical manipulation of many closely-spaced nanoparticles and&#160;limits the&#160;resolution of optical-mechanical systems. Furthermore, manipulation using focused beams requires beam-forming or steering optics, which can be very bulky and expensive. To address these limitations in the system scalability of conventional optical trapping our lab has devised an alternative technique which utilizes near-field optics to move particles across a chip. Instead of focusing laser beams in the far-field, the optical near field of plasmonic resonators produces the necessary local optical intensity enhancement to overcome the restrictions of diffraction and manipulate particles at higher resolution. Closely-spaced resonators produce strong optical traps which can be addressed to mediate the hand-off of particles from one to the next in a conveyor-belt-like fashion. Here, we describe how to design and produce a conveyor belt using a gold surface patterned with plasmonic C-shaped resonators and how to operate it with polarized laser light to achieve super-resolution nanoparticle manipulation and transport. The nano-optical conveyor belt chip can be produced using lithography techniques and easily packaged and distributed.

The scalability and resolution of conventional optical manipulation techniques are limited by diffraction. We circumvent the diffraction limit and describe a method of optically transporting nanoparticles across a chip using a gold surface patterned with a path of closely spaced C-shaped plasmonic resonators.

一个纳米光输送带的制作和操作

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

使用双光镊和微流体进行单分子研究

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

生物化学

Optical Biosensing

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence,&#160;or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

Types of Optical Biosensing; Surface Plasmon Resonance SPR

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence, or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

光学传感

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable ...