作者感谢 Prof. y Ozaki (Kwansei 学院大学) 和 Dr. (国家高等工业科技研究院) 对这项工作进行了卓有成效的讨论。这项工作得到了教育、文化、体育、科学和技术部 (No. 16K05671) 的 KAKENHI (补助金) 的支持。

1. 样品制备
<ol><li>
制备银胶体纳米粒子20<ol>
<li>制备银胶体纳米粒子, 溶解0.030 克硝酸银和0.030 克柠檬酸三钠, 在150毫升水中的200毫升圆形底烧瓶。</li>
		<li>将烧瓶与回流 (Dimroth) 冷凝器结合。</li>
		<li>用磁力搅拌器和搅拌棒在烧瓶中搅拌溶液。然后, 在150° c 的油浴中, 在烧瓶中加热搅拌溶液60分钟。 注: 溶液将变成黄色, 然后是乳白色的灰色。</li>
		<li>在室温下冷却悬浮液, 并保持在冰箱内用铝箔覆盖的烧瓶中的悬浮。 注意: 在此点可以暂停协议。在一个月内, 用胶体纳米微粒储存在冰箱里。</li>
	</ol>
</li>
	<li>
多色闪烁发射样品的制备11<ol>
<li>要准备一个显微镜幻灯片, 用手用肥皂冲洗玻璃盘子, 并用清水冲洗。</li>
		<li>在玻璃板上加入0.1% 多聚 l-赖氨酸水溶液, 用鼓风机将溶液取出。</li>
		<li>将银胶体悬浮液添加到玻璃板上, 然后用鼓风机将悬浮液除去。</li>
		<li>用液体阻挡笔将玻璃板上的拖放区域括起来。</li>
		<li>将蒸馏水滴在玻璃板上, 用另一玻璃板盖上, 以制造显微镜滑动, 防止水蒸发。</li>
	</ol>
</li>
	<li>
为单调的彩色闪烁 SERS 进行样品制备7,8,9,10<ol>
<li>要准备一个显微镜幻灯片, 用手用肥皂冲洗玻璃盘子, 并用清水冲洗。</li>
		<li>将银胶体悬浮液与 thiacyanine 或 thiacarbocyanine 染料 (分别为25或4µM) 和氯化钠 (10 毫米) 水溶液混合, 体积比为 2:1: 1。</li>
		<li>将样品悬浮液放在玻璃板上, 然后用鼓风机将悬挂装置卸下。</li>
		<li>用液体阻挡笔将玻璃板上的拖放区域括起来。</li>
		<li>将氯化钠 (1 米) 的水溶液滴在玻璃板上, 使银纳米颗粒固定, 并用另一玻璃板盖住, 以制造显微镜滑动板, 防止溶液蒸发。</li>
	</ol>
</li>
</ol>2. 闪光银纳米粒子的观察
<ol><li>
样品照明
	<ol>
<li>将使用协议1.2 或1.3 的样品玻璃板放在倒置显微镜的舞台上。</li>
		<li>通过暗场冷凝器照亮样品玻璃板, 用物镜 (60X) 在玻璃板上聚焦各种彩色斑点 (蓝色、绿色、黄色和红色)。</li>
		<li>用衰减光束照射样品玻璃板, 其相对于样品表面的角度为30°, 从二极管泵浦固态 (DPSS) continuous-wave (cw) 激光器通过干涉滤光片。</li>
		<li>利用激光照射在同色的环境中观察银粒子为单调的彩色斑点, 将激光照射区移至视图中心, 并通过调整 z 方向上的舞台来聚焦在玻璃板上的斑点上。</li>
	</ol>
</li>
	<li>
闪烁的观察
	<ol>
<li>在物镜后插入一个 long-pass 过滤器, 并通过干涉滤光片, 在30°相对于样品表面的情况下, 用 DPSS 的连续激光光束照射样品玻璃板。</li>
		<li>通过在 x 和 y 方向移动舞台, 查找图 1中显示的闪烁点 (也请参见辅助材料中的图 S1 )。</li>
		<li>用倒置显微镜记录闪烁斑点的视频, 耦合到一个冷却的数字电荷耦合器件 (CCD) 相机, 它的时间分辨率为 61-120 毫秒, 为20分钟。</li>
	</ol>
</li>
</ol>3. 闪光 SERS 的分析
<ol><li>
从视频中推导时间剖面
	<ol>
<li>在控制 CCD 摄像机的软件中, 打开视频文件。</li>
		<li>若要选择闪烁的斑点和暗区, 请拖动单独覆盖视频图像中有斑点的区域的区域。</li>
		<li>若要从视频中的闪烁点和暗区域派生信号强度时间配置文件, 请在分析中选择时间分析, 然后在时间分析窗口中单击计算。</li>
		<li>将数据另存为文本文件。</li>
	</ol>
</li>
	<li>
时间剖面分析
	<ol>
<li>通过从暗区和/或与多项式函数的拟合中减去时间轮廓来拼合时间模板的基线, 如图 2A和2B所示。</li>
		<li>计算由大约2000点、 I基和基线强度标准偏差 (σ) 组成的平均基线强度, 如图 2C和2D中所示。</li>
		<li>使用比I基+ 3 的阈值更大的强度来区分明亮事件和暗事件, 并记录每个事件的持续时间。例如, 在图 3中, 将事件从0记录为黑色事件 (其持续时间为t = 3.5476 秒), 并将事件从3.5476 个到4.0981 秒记录为明亮事件 (持续时间为t = 0.5505 秒)。重复过程, 如表 1所示。</li>
		<li>按照表 2的第一和第二行中的表示, 计算每个持续时间内的亮和暗事件数。</li>
		<li>对每个工期的事件数求和, 但事件少于持续时间t。例如, 在表 2的第二和第三行中表示, 对每个持续时间的事件数进行求和 ( t = 0.0612 秒的事件除外) 为 41 + 18 + 9 +..。结果等于t = 0.1223 s 的求和,即103。</li>
		<li>按每个工期划分总结, 并对其进行规范化。例如, 如表 2所示, 将持续时间的总和t = 0.0612 s 除以持续时间0.0612 秒。结果是3351.5791。然后, 将结果除以表 2中第四行中的结果总数。概率分布被导出为0.64494。</li>
		<li>将明亮事件的概率分布绘制为Pon(t), 并将其持续时间t在对数对数图中, 并适合日志10P(t) 的日志10()<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">在上为特定的闪烁点推断幂定律指数α。<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">如果Pon(t) 是由安装的, 则拟合的行将在P(t)上的小数值上偏离图形, 如图 4A中的虚线所示。</li>
		<li>按日志10将暗事件的概率分布绘制为P关(t), 并将其持续时间t在对数对数图中, 并适合日志10P关闭(t)。从相同的闪烁点推断出幂律指数α <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> off和截断时间τ 。如果Poff(t) 是由安装的, 则拟合的曲线将从位于<img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> </img> P关闭(t) 的小值中偏离图形。</li>
		<li>对视频中的其他闪烁点重复3.2.1 到3.2.8。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Qian, X. M., Nie, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+and+single-nanoparticle+SERS:+from+fundamental+mechanisms+to+biomedical+applications.">Single-molecule and single-nanoparticle SERS: from fundamental mechanisms to biomedical applications.</a> Chem. Soc. Rev. 37, 912-920 (2008).</li><li>Pieczonka, N. P. W., Aroca, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+analysis+by+surfaced-enhanced+Raman+scattering.">Single molecule analysis by surfaced-enhanced Raman scattering.</a> Chem. Soc. Rev. 37, 946-954 (2008).</li><li>Kneipp, J., Kneipp, H., Kneipp, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SERS+-a+single-molecule+and+nanoscale+tool+for+bioanalytics.">SERS -a single-molecule and nanoscale tool for bioanalytics.</a> Chem. Soc. Rev. 37, 1052-1060 (2008).</li><li>Kitahama, Y., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blinking+SERS+by+a+power+law+with+an+exponential+function.">Analysis of blinking SERS by a power law with an exponential function.</a> Frontiers of Surface-Enhanced Raman Scattering: Single-Nanoparticles and Single Cells. , (2014).</li><li>Kitahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncated+Power+Law+Analysis+of+Blinking+SERS.">Truncated Power Law Analysis of Blinking SERS.</a> Frontiers of Plasmon Enhanced Spectroscopy Volume 1 (ACS Symposium series Vol. 1245). , (2016).</li><li>Bizzarri, A. R., Cannistraro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L&#233;vy+Statistics+of+Vibrational+Mode+Fluctuations+of+Single+Molecules+from+Surface-Enhanced+Raman+Scattering.">L&#233;vy Statistics of Vibrational Mode Fluctuations of Single Molecules from Surface-Enhanced Raman Scattering.</a> Phys. Rev. Lett. 94, 068303 (2005).</li><li>Kitahama, Y., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power-law+analysis+of+surface-plasmon-enhanced+electromagnetic+field+dependence+of+blinking+SERS+of+thiacyanine+or+thiacarbocyanine+adsorbed+on+single+silver+nanoaggregates.">Power-law analysis of surface-plasmon-enhanced electromagnetic field dependence of blinking SERS of thiacyanine or thiacarbocyanine adsorbed on single silver nanoaggregates.</a> Phys. Chem. Chem. Phys. 13, 7439-7448 (2011).</li><li>Kitahama, Y., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+excitation+laser+intensity+dependence+of+blinking+SERRS+of+thiacarbocyanine+adsorbed+on+single+silver+nanoaggregates+by+using+a+power+law+with+an+exponential+function.">Analysis of excitation laser intensity dependence of blinking SERRS of thiacarbocyanine adsorbed on single silver nanoaggregates by using a power law with an exponential function.</a> Chem. Commun. 47, 3888-3890 (2011).</li><li>Kitahama, Y., Enogaki, A., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncated+power+law+analysis+of+blinking+SERS+of+thiacyanine+molecules+adsorbed+on+single+silver+nanoaggregates+by+excitation+at+various+wavelengths.">Truncated power law analysis of blinking SERS of thiacyanine molecules adsorbed on single silver nanoaggregates by excitation at various wavelengths.</a> J. Phys. Chem. C. 117, 9397-9403 (2013).</li><li>Kitahama, Y., Araki, D., Yamamoto, Y. S., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+behaviour+of+molecules+in+dark+SERS+state+on+colloidal+Ag+nanoparticles+estimated+by+truncated+power+law+analysis+of+blinking+SERS.">Different behaviour of molecules in dark SERS state on colloidal Ag nanoparticles estimated by truncated power law analysis of blinking SERS.</a> Phys. Chem. Chem. Phys. 17, 21204-21210 (2015).</li><li>Kitahama, Y., Nagahiro, T., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blinking+from+multicoloured+SERS-active+Ag+colloidal+nanoaggregates+with+poly-L-lysine+via+truncated+power+law.">Analysis of blinking from multicoloured SERS-active Ag colloidal nanoaggregates with poly-L-lysine via truncated power law.</a> J. Raman. Spectrosc. 48, 570-577 (2017).</li><li>Habuchi, S., 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=Single-Molecule+Surface+Enhanced+Resonance+Raman+Spectroscopy+of+the+Enhanced+Green+Fluorescent+Protein.">Single-Molecule Surface Enhanced Resonance Raman Spectroscopy of the Enhanced Green Fluorescent Protein.</a> J. Am. Chem. Soc. 125, 8446-8447 (2003).</li><li>Weiss, A., Haran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-Dependent+Single-Molecule+Raman+Scattering+as+a+Probe+of+Surface+Dynamics.">Time-Dependent Single-Molecule Raman Scattering as a Probe of Surface Dynamics.</a> J. Phys. Chem. B. 105, 12348-12354 (2001).</li><li>Emory, S. R., Jensen, R. A., Wenda, T., Han, M., Nie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-examining+the+origins+of+spectral+blinking+in+single-molecule+and+single-nanoparticle+SERS.">Re-examining the origins of spectral blinking in single-molecule and single-nanoparticle SERS.</a> Faraday Discuss. 132, 249-259 (2006).</li><li>Itoh, T., Iga, M., Tamaru, H., Yoshida, K., Biju, V., Ishikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+blinking+in+surface+enhanced+resonance+Raman+scattering+and+fluorescence+by+electromagnetic+mechanism.">Quantitative evaluation of blinking in surface enhanced resonance Raman scattering and fluorescence by electromagnetic mechanism.</a> J. Chem. Phys. 136, 024703 (2012).</li><li>Willets, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+of+SERS+hot+spots.">Super-resolution imaging of SERS hot spots.</a> Chem. Soc. Rev. 43, 3854-3864 (2014).</li><li>Dieringer, J. A., Lettan, R. B., Scheidt, K. A., Van Duyne, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Frequency+Domain+Existence+Proof+of+Single-Molecule+Surface-Enhanced+Raman+Spectroscopy.">A Frequency Domain Existence Proof of Single-Molecule Surface-Enhanced Raman Spectroscopy.</a> J. Am. Chem. Soc. 129, 16249-16256 (2007).</li><li>Cichos, F., von Borczyskowski, C., Orrit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power-law+intermittency+of+single+emitters.">Power-law intermittency of single emitters.</a> Curr. Opin. Colloid Interface Sci. 12, 272-284 (2007).</li><li>Tang, J., Marcus, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fluorescence+blinking+in+semiconductor+nanocrystal+quantum+dots.">Mechanisms of fluorescence blinking in semiconductor nanocrystal quantum dots.</a> J. Chem. Phys. 123, 054704 (2005).</li><li>Lee, P. C., Meisel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+surface-enhanced+Raman+of+dyes+on+silver+and+gold+sols.">Adsorption and surface-enhanced Raman of dyes on silver and gold sols.</a> J. Phys. Chem. 86, 3391-3395 (1982).</li><li>Krichevsky, O., Bonnet, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+the+technique+and+its+applications.">Fluorescence correlation spectroscopy: the technique and its applications.</a> Rep. Prog. Phys. 65, 251-297 (2002).</li><li>Hess, S. T., Huang, S., Heikal, A. A., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+Chemical+Applications+of+Fluorescence+Correlation+Spectroscopy:+A+Review.">Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A Review.</a> Biochemistry. 41, 697-705 (2002).</li></ol>

作者没有什么要透露的。

从银 nanoaggregate 结, SERS 被发射。因此, 我们需要准备粒子而不是胶体纳米粒子, 这是被柠檬酸阴离子覆盖。银聚合体是由添加多聚 l-赖氨酸所产生的盐析效应形成的, 它具有-NH3+ , 是来自氯化钠的 SERS、Na+阳离子的来源, 如补充材料的图 S2所示。此外, 为了照亮宽区的许多斑点, 不聚焦的激光光束是通过一个不附着在显微镜上的透镜, 相对于样品表面, 以30°的角度交付的。有可能观察区域没有被照亮。我们调整和移动激光区域以高放大率照亮观察区域。经过这一优化, 单调的彩色斑点是可见的在同色的环境。这些都是用于闪烁 SERS 观察的关键步骤。
在此, 讨论了幂律分析中需要注意的问题。首先, 明暗事件定义的阈值会影响闪烁的分析。当阈值增加时, 幂律指数和截断次数也趋于增加,4,5,9。当指数 (αon和αoff) 和截断时间呈现不同的趋势时, 可以发现闪烁的 SERS 的依赖性。其次, 较小幂律指数表示在对数对数图中幂律所给出的直线的陡斜率, 表示亮或暗事件7的长持续时间的较低概率。因为明亮的事件不能持续很长的时间, 所以在图中绘制明亮事件的点数比暗事件的少。然后, αon值往往小于αoff, 值7, 不同于从单个量子点 (αoff = α在=-1.5)18的闪烁。第三, 指数只会稍微大于-110, 因为概率分布是通过以下公式给出的:
<img alt="Equation 7" src="/files/ftp_upload/56729/56729eq7.jpg">,
其分子在中期 (派生自协议 3.2.5; 请参见表 2的第三行) 倾向于减少 t 的较长的持续时间,因为即使更长的持续时间的光明和黑暗事件的数量往往会因以下事实而减少分子随机移动, 几乎不能停留在一个发射状态或放射状态 (nanoaggregate 的交界处) 很长一段时间, 在第二行表 2中表示。幂定律指数α =-1.5 或-1, 可以从分子随机行走于银表面一-或 two-dimensionally, 分别为4,5,18的事实派生。相比之下, 截断时间缩短的分子随机步行和/或更高的能量屏障从发射到放射状态4,5,19。我们注意到, 概率分布不能用幂律来再现的事件的百分比是重要的数据9,10, 因为复制失败源自很长截断时间。
在以前的研究中12,13,14, 自相关函数也用于闪烁的 SERS。自相关函数, 这是用于荧光相关光谱学, 代表扩散系数和浓度的荧光分子进出的重点领域21,22。然而, 对于闪烁的 SERS, 没有简单的函数可以重现自相关函数14。这意味着一个复杂的过程, 闪烁的 SERS, 因为自相关函数可以确定一些周期性。在另一种定量分析中, 从信号的时间波形15中导出了规范化的标准偏差分数。大比分表明, 总强度不稳定。这些分析方法可能适用于少数分子的行为, 而不是单个分子的行为。此外, 明亮事件的平均工期用于分析闪烁的 SERS4,14。这些可以揭示分子在增强的 EM 场中的行为, 类似于 SERS16的超分辨率成像。然而, 黑暗事件的平均值不能得到;即, 由单个银粒子的暗 sers 事件的总持续时间是通过增加事件4的次数而减少的, 而明亮的 sers 事件的总工期则增加了。因此, 只有光 SERS 事件的分子行为才能被这些技术所研究。通过使用幂定律分析, 另一方面, 黑暗状态的分子行为 (即在银表面上, 除了银粒子的接合点) 可以根据幂律指数αoff和截断时间10来估计.这与以前的技术有很大的不同。
为了确认该闪烁是由 SERS 引起的, 光谱是从一个 nanoaggregate 的中心, 通过一个针孔来测量的, 如在补充材料的图 S3中所示。但是, 它不是从所有闪烁的点7、8、9、10、11中进行测量的。对于多聚 l-赖氨酸的11, 在长波区域中闪烁的原因不是 sers, 而是表面增强的荧光, 这也来源于增强的 EM 领域, 如 sers。此外, 有争议的是连接的波动频谱与闪烁。这是目前技术的一个局限。
在未来, 应根据每个峰值来分析闪烁的 SERS。即幂律分析应与频谱成像相结合。通过使用双分析物技术17, 在混合分析中观察到独特的振动特征, 分子 SERS 的起源得到了证实。然而, 由于时间的推移, 分子行为不能被显著的研究。最近, 通过每个光过滤器和幂定律分析11, 通过观察每个闪烁点, 从截断时间上检测出每个源的不同行为。

表面增强拉曼散射 (SERS) 是高灵敏度的拉曼光谱从一个高贵的金属表面。由于拉曼光谱提供了基于尖峰位置的分子结构的详细信息, 通过分子中官能团的振动模式, 可以研究金属表面上的单个分子的信息使用 SERS1,2,3。从银 nanoaggregate 与吸附在分子水平, 一个闪烁的信号被 观察1,2,3,45,7,8,9,10,11,12,13,14,15, 16, 频谱波动1,2,3,4,5,67,8, 9,10,11,12,13,14。眨眼可以由一个单一的分子, 随机进出一个增强电磁 (EM) 领域在一个纳米银 nanoaggregate 交界处。因此, 闪烁被认为是分子检测的简单证据, 与使用 SERS 强度的泊松分布和双分析物2、3、17的技术相比。然而, 闪烁和波动频谱的详细机制, 可能强烈依赖于分子行为在 Ag 表面, 仍然是有争议的。
在先前的研究中, 用自相关函数分析了闪烁的 SERS, 它可以计算在增强的 EM 场中进出的分子的扩散系数和浓度12,13,14.此外, 一个标准化的标准偏差评分, 代表了不稳定性的总强度, 是从信号的时间剖面的15。然而, 这些分析方法可能是基于一些分子的行为。相比之下, 在一个超分辨率成像的闪烁 SERS, 分子行为在增强 EM 领域可以识别16。但是, 这些技术只能在增强的 EM 域中获得这些参数。单个分子在广泛范围内的随机行为 (例如, 在闪烁的 SERS 中) 可以表示为幂定律, 而不是平均4,5,6,7,8 ,9,10,11, 类似于单个半导体量子点 (数字)18,19中闪烁的荧光。通过使用幂律分析4、5、6、7、8、91011, 分子行为可以估计在明亮状态 (在增强的 EM 领域) 和黑暗状态10;那是, 分子的行为在整个银色表面可以估计。
对于此技术, 银胶体粒子使用4,5,6,,8,9,10,11。这些粒子显示各种局部的表面等离子共振 (LSPR) 波段, 强烈影响增强的电磁场时, 他们是在一定的波长兴奋。因此, SERS 活性银纳米粒子存在于胶体悬浮液中, 并能立即获得一些数据。在简单的纳米结构的情况下, 有特定的大小, 形状和安排, LSPR 依赖的 SERS 闪烁可以隐藏其他依赖性7;也就是说, 如果使用了 LSPR 的好的或坏的纳米结构, 则参数将是恒定的, 因此其他的依赖将被隐藏。幂律分析已被用于发现从银胶体粒子的各种依赖的闪光 SERS, 4,5,6,7,89,10,11。

<table border="0" cellpadding="0" cellspacing="0" style="width:639px;" width="638">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Silver nitrate, 99.8%</td>
			<td style="width:83px;">Wako</td>
			<td style="width:119px;">194-00832</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Trisodium citrate dihydrate, 99. %</td>
			<td style="width:83px;">Wako</td>
			<td style="width:119px;">191-01785</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Poly-L-lysine aqueous solution, 0.1%</td>
			<td style="width:83px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="116">
			<td height="116" style="height:116px;width:227px;">3,3&#39;-disulfopropylthiacyanine triethylamine</td>
			<td style="width:83px;">Hayashibara Biochemical Laboratories</td>
			<td style="width:119px;">NK-2703</td>
			<td>a kind of thiacyanine dyes</td>
		</tr>
		<tr height="116">
			<td height="116" style="height:116px;width:227px;">3,3&#39;-diethyl-5,5&#39;-dichloro-9-methylthiacarbocyanine iodine salt</td>
			<td style="width:83px;">Hayashibara Biochemical Laboratories</td>
			<td style="width:119px;">SMP-9</td>
			<td>a kind of thiacarobocyanine dyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Sodium chloride, 99.5%</td>
			<td style="width:83px;">Wako</td>
			<td style="width:119px;">191-01665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Dimroth condenser</td>
			<td style="width:83px;">Iwaki</td>
			<td style="width:119px;">61-9722-22</td>
			<td>perchased from AS ONE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Magnetic stirrer</td>
			<td style="width:83px;">Corning</td>
			<td style="width:119px;">DC-420D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Oil bath</td>
			<td style="width:83px;">Advantech</td>
			<td style="width:119px;">OS-220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Glass plate</td>
			<td style="width:83px;">Matsunami</td>
			<td style="width:119px;">S-1112</td>
			<td>Microscope slide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Blower</td>
			<td style="width:83px;">Hozan</td>
			<td style="width:119px;">Z-288</td>
			<td>Air duster</td>
		</tr>
		<tr height="116">
			<td height="116" style="height:116px;width:227px;">Liquid blocker pen</td>
			<td style="width:83px;">Daido Sangyo</td>
			<td style="width:119px;"></td>
			<td style="width:211px;">LIQUID BLOCKER (Super Pap Pen). Ready-to-use hydrophobic barrier pen designed for immunohistochemistry applications</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Inverted microscope</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:119px;">IX-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Objective lens</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:119px;">LCPlanFl 60&#215;</td>
			<td>NA 0.7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Dark field condenser</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:119px;">U-DCD</td>
			<td style="width:211px;">NA 0.8&#8211;0.92</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Cooled digital CCD camera</td>
			<td style="width:83px;">Hamamatsu</td>
			<td style="width:119px;">ORCA-AG</td>
			<td style="width:211px;">controlled by software Aqua Cosmos</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:227px;">Software for the cooled digital CCD camera</td>
			<td style="width:83px;">Hamamatsu</td>
			<td style="width:119px;">AquaCosmos</td>
			<td style="width:211px;">used for also derivation of the time-profiles from the blinking spots in the video&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Color CCD camera</td>
			<td style="width:83px;">ELMO</td>
			<td style="width:119px;">TNC-C920</td>
			<td>not used for analysis</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:227px;">DPSS laser</td>
			<td style="width:83px;">RGB laser system</td>
			<td style="width:119px;">NovaPro532-75</td>
			<td style="width:211px;">&#955; = 532 nm; 
			60 mW (corresponds to a power density of 600 W/cm2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Interference filter</td>
			<td style="width:83px;">Semrock</td>
			<td style="width:119px;">LL01-532-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Long pass filter</td>
			<td style="width:83px;">Semrock</td>
			<td style="width:119px;">BLP01-532R-25</td>
			<td></td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:227px;">Software for the distinguishment and counting of the bright/dark events</td>
			<td style="width:83px;">home-maid</td>
			<td style="width:119px;"></td>
			<td>programmed by C++</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Software for the fitting by a power law</td>
			<td style="width:83px;">LightStone</td>
			<td style="width:119px;">Origin6.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

observation and analysis of blinking surface-enhanced raman scattering

从一个单一的分子在银 nanoaggregate 结, 闪烁的表面增强拉曼散射 (SERS) 观察。本文介绍了如何制备 SERS 活性银 nanoaggregate, 在显微图像中记录某些闪烁点的视频, 并分析闪烁的统计数据。在这个分析中, 幂律再现了明亮事件相对于其持续时间的概率分布。黑暗事件的概率分布由幂律和指数函数拟合。幂定律的参数代表了光明和黑暗状态的分子行为。随机游走模型和分子在整个银色表面的速度可以估计。即使使用平均值、自相关函数和超分辨率 SERS 成像, 也难以估计。在未来, 幂律分析应与频谱成像相结合, 因为这种分析方法不能单独证实闪烁的起源。

从1.2 号协议制备的多聚 l-赖氨酸银粒子, 观察到了 SERS 和表面增强荧光的多色闪烁斑点, 如图 111所示。相比之下, 由 1.37、8、9、10所制备的染料分子, 在银粒子上观察到来自 SERS 的单调彩色闪烁点。有两种类型的 "阴性" 结果: 连续的结果, 或结果, 没有 SERS 的观察。前者和后者的结果可能是由高或低浓度的分子在胶体银表面, 分别。
来自单个银 nanoaggregate 的信号在不同的时间显示各种强度, 如图 2B所示。这是不同的闪烁荧光的一个单一的量子。荧光强度的直方图显示两个不同的峰值, 它们代表光明和黑暗状态18。在较长范围内的时间分布类似于在短范围内的扩展, 如图 34。这被称为 "自相似" 或 "分形";也就是说, 如果扩展了对象的长度尺度, 它们的特征就会被认为是相似的。
在对数对数图中, 明亮和暗事件的概率分布分别以直线和曲线的形式绘制, 如图 4所示 (相对于单个量子数的闪烁)19。在图中, 直线的斜率对应幂律指数。相反, 较短的截断时间是从一个事实, 即黑暗状态的幂律被截断在较短的尾巴。对于暗 SERS 事件, 概率分布有时是由幂律而不是指数函数拟合的。即, 很长的截断时间有很大的错误, 有时会导出9,10。然而, 具有指数函数的幂律不能重现暗 SERS 事件的概率分布, 这并不是一个 "负" 结果。
幂定律指数α开/关和截断时间τ从单个银纳米粒子推断出不同的值, 如 图 5所示。从多个幂律指数中, 推导出一个标准误差的平均值, 并与不同条件下的其他值进行比较。在截断时间的情况下, 中位数而不是平均值可能适合比较。幸运的是, 大量的数据可以从几个闪烁的视频中收集到, 因为在视频中可以同时观察到十几个闪烁的斑点。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56729/56729fig1.jpg"> 图 1:有代表性的闪烁的 SERS 图像.多色闪烁斑点从银粒子观察与聚 l-赖氨酸。缩放条 = 10 µm。这是采取倒置显微镜耦合到彩色 CCD 相机通过一个长的通行证过滤器 (见相应的视频电影在图 S1的补充材料)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/56729fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56729/56729fig2.jpg"> 图 2: (A) 来自闪烁点的信号强度的代表时间波形.(B) 通过多项式函数从暗区和/或管接头中减去时间剖面, 使基线已被拼合的时间配置文件。转载的许可, 从皇家化学学会8。(C) 放大 (B) 中的正方形, 即时间模板的基线。(D) 基线点强度的示意图密度。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/56729fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56729/56729fig3.jpg"> 图 3:来自闪烁点的信号强度的代表时间轮廓和明暗事件定义的阈值 (水平线)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/56729fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56729/56729fig4.jpg"> 图 4:针对其工期绘制的闪烁点的代表性概率分布。(a) 在对数对数图中针对其工期绘制的亮事件的概率分布。<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">实线 <img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">和虚线是使用公式进行拟合的结果, 它们分别为日志10pon(t) = 日志10() 和p(t) =。(B) 在对数对数图中针对其工期绘制的暗事件的概率分布。它们可以被拟合为幂律具有指数函数的曲线。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/56729fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56729/56729fig5.jpg"> 图 5:来自幂律的参数的代表性直方图。(A) 幂定律指数对亮事件的直方图。(B) 暗事件幂律指数的直方图。(C) 幂律中截断时间的直方图, 用于暗事件的指数函数。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/56729fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/56729/56729supfig1.jpg"> 图 S1: 具有代表性的闪烁 SERS 的电影.多色闪烁斑点从银粒子观察与聚 l-赖氨酸。这包括一个面积50µm x 40 µm, 并采取了倒置显微镜耦合到彩色 CCD 相机通过一个长传球过滤器。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/Fig1.mov" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Supplemental Figure 2" src="/files/ftp_upload/56729/56729supfig2.jpg"> 图 S2: 具有代表性的扫描电子显微镜图像的银 nanoaggregate 形成的多聚 l-赖氨酸或氯化钠.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/56729supfig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplemental Figure 3" src="/files/ftp_upload/56729/56729supfig3.jpg"> 图 S3: thiacarbocyanine 粉末的常规拉曼光谱, 以及具有代表性的具有 thiacarbocyanine 的单银 nanoaggregate 的时间波动的 SERS 谱.转载的许可, 从皇家化学学会8。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56729/56729supfig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>事件</td>
			<td>开始时间/秒</td>
			<td>结束时间/秒</td>
			<td>持续时间/秒</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>0.0000</td>
			<td>3.5476</td>
			<td>3.5476</td>
		</tr>
<tr>
<td>明亮</td>
			<td>3.5476</td>
			<td>4.0981</td>
			<td>0.5505</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>4.0981</td>
			<td>5.8720</td>
			<td>1.7738</td>
		</tr>
<tr>
<td>明亮</td>
			<td>5.8720</td>
			<td>5.9331</td>
			<td>0.0612</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>5.9331</td>
			<td>6.3613</td>
			<td>0.4282</td>
		</tr>
<tr>
<td>明亮</td>
			<td>6.3613</td>
			<td>6.4836</td>
			<td>0.1223</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>6.4836</td>
			<td>6.6671</td>
			<td>0.1835</td>
		</tr>
<tr>
<td>明亮</td>
			<td>6.6671</td>
			<td>6.7895</td>
			<td>0.1223</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>6.7895</td>
			<td>7.0341</td>
			<td>0.2447</td>
		</tr>
<tr>
<td>明亮</td>
			<td>7.0341</td>
			<td>7.0953</td>
			<td>0.0612</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>7.0953</td>
			<td>8.3798</td>
			<td>1.2845</td>
		</tr>
<tr>
<td>明亮</td>
			<td>8.3798</td>
			<td>8.4409</td>
			<td>0.0612</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>8.4409</td>
			<td>8.6856</td>
			<td>0.2447</td>
		</tr>
<tr>
<td>明亮</td>
			<td>8.6856</td>
			<td>8.7468</td>
			<td>0.0612</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>8.7468</td>
			<td>9.6643</td>
			<td>0.9175</td>
		</tr>
<tr>
<td>明亮</td>
			<td>9.6643</td>
			<td>9.9089</td>
			<td>0.2447</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>9.9089</td>
			<td>9.9701</td>
			<td>0.0612</td>
		</tr>
<tr>
<td>明亮</td>
			<td>9.9701</td>
			<td>10.3371</td>
			<td>0.3670</td>
		</tr>
<tr>
<td>黑暗</td>
			<td>10.3371</td>
			<td>10.3983</td>
			<td>0.0612</td>
		</tr>
</tbody></table>表 1: 暗或亮事件、事件开始时间、事件结束时间和事件持续时间的代表性表.这些派生自图 3。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>持续时间/秒</td>
			<td>笑的事件</td>
			<td>求和</td>
			<td>(总和)/(持续时间)</td>
			<td>概率分布/s-1</td>
		</tr>
<tr>
<td>0.0612</td>
			<td>102</td>
			<td>205</td>
			<td>3351.5791</td>
			<td>0.64494</td>
		</tr>
<tr>
<td>0.1223</td>
			<td>41</td>
			<td>103</td>
			<td>841.9821</td>
			<td>0.16202</td>
		</tr>
<tr>
<td>0.1835</td>
			<td>18</td>
			<td>62</td>
			<td>337.8828</td>
			<td>0.06502</td>
		</tr>
<tr>
<td>0.2447</td>
			<td>9</td>
			<td>44</td>
			<td>179.8408</td>
			<td>0.03461</td>
		</tr>
<tr>
<td>0.3058</td>
			<td>4</td>
			<td>35</td>
			<td>114.4442</td>
			<td>0.02202</td>
		</tr>
<tr>
<td>0.3670</td>
			<td>3</td>
			<td>31</td>
			<td>84.4707</td>
			<td>0.01626</td>
		</tr>
<tr>
<td>0.4282</td>
			<td>3</td>
			<td>28</td>
			<td>65.3967</td>
			<td>0.01258</td>
		</tr>
<tr>
<td>0.4893</td>
			<td>4</td>
			<td>25</td>
			<td>51.0911</td>
			<td>0.00983</td>
		</tr>
<tr>
<td>0.5505</td>
			<td>1</td>
			<td>21</td>
			<td>38.1481</td>
			<td>0.00734</td>
		</tr>
<tr>
<td>0.6117</td>
			<td>1</td>
			<td>20</td>
			<td>32.6983</td>
			<td>0.00629</td>
		</tr>
<tr>
<td>0.6728</td>
			<td>5</td>
			<td>19</td>
			<td>28.2395</td>
			<td>0.00543</td>
		</tr>
<tr>
<td>0.7340</td>
			<td>2</td>
			<td>14</td>
			<td>19.0740</td>
			<td>0.00367</td>
		</tr>
<tr>
<td>0.9786</td>
			<td>1</td>
			<td>12</td>
			<td>12.2619</td>
			<td>0.00236</td>
		</tr>
<tr>
<td>1.0398</td>
			<td>1</td>
			<td>11</td>
			<td>10.5789</td>
			<td>0.00204</td>
		</tr>
<tr>
<td>1.1621</td>
			<td>2</td>
			<td>10</td>
			<td>8.6048</td>
			<td>0.00166</td>
		</tr>
<tr>
<td>1.3456</td>
			<td>1</td>
			<td>8</td>
			<td>5.9452</td>
			<td>0.00114</td>
		</tr>
<tr>
<td>1.4068</td>
			<td>1</td>
			<td>7</td>
			<td>4.9758</td>
			<td>0.00096</td>
		</tr>
<tr>
<td>1.9573</td>
			<td>1</td>
			<td>6</td>
			<td>3.0655</td>
			<td>0.00059</td>
		</tr>
<tr>
<td>2.0796</td>
			<td>1</td>
			<td>5</td>
			<td>2.4043</td>
			<td>0.00046</td>
		</tr>
<tr>
<td>2.2631</td>
			<td>1</td>
			<td>4</td>
			<td>1.7675</td>
			<td>0.00034</td>
		</tr>
<tr>
<td>2.4466</td>
			<td>1</td>
			<td>3</td>
			<td>1.2262</td>
			<td>0.00024</td>
		</tr>
<tr>
<td>2.8136</td>
			<td>1</td>
			<td>2</td>
			<td>0.7108</td>
			<td>0.00014</td>
		</tr>
<tr>
<td>2.9359</td>
			<td>1</td>
			<td>1</td>
			<td>0.3406</td>
			<td>0.00007</td>
		</tr>
</tbody></table>表 2: 代表性的工期表、每个工期的事件数、较长工期的事件数求和、总结除以每个工期以及它们的规范化概率分布。

Watch this Scientific Journal Video about Observation and Analysis of Blinking Surface-enhanced Raman Scattering at JoVE.com

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

该协议描述了由于在银色表面上使用幂定律的单个分子的随机游走而引起的表面增强拉曼散射的分析。

闪烁表面增强拉曼散射的观测与分析

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

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7.3K 次观看.  Kwansei Gakuin University. 该协议描述了由于在银色表面上使用幂定律的单个分子的随机游走而引起的表面增强拉曼散射的分析。

视频: Observation and Analysis of Blinking Surface-enhanced Raman Scattering

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

基于过滤器的表面增强拉曼光谱检测方法的化学污染物快速检测

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

科研

化学

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

生物聚合物气凝胶制备使用绿色溶剂

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

通过动态光散射显微镜混浊解决方案粒度分布的测定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

A Novel Technique for Raman Analysis of Highly Radioactive Samples Using Any Standard Micro-Raman Spectrometer

A novel approach for the Raman measurement of nuclear materials is reported in this paper. It consists of the enclosure of the radioactive sample in a tight capsule that isolates the material from the atmosphere. The capsule can optionally be filled with a chosen gas pressurized up to 20 bars. The micro-Raman measurement is performed through an optical-grade quartz window. This technique permits accurate Raman measurements with no need for the spectrometer to be enclosed in an alpha-tight containment. It therefore allows the use of all options of the Raman spectrometer, like multi-wavelength laser excitation, different polarizations, and single or triple spectrometer modes. Some examples of measurements are shown and discussed. First, some spectral features of a highly radioactive americium oxide sample (AmO2) are presented. Then, we report the Raman spectra of neptunium oxide (NpO2) samples, the interpretation of which is greatly improved by employing three different excitation wavelengths, 17O doping, and a triple mode configuration to measure the anti-stokes Raman lines. This last feature also allows the estimation of the sample surface temperature. Finally, data that were measured on a sample from Chernobyl lava, where phases are identified by Raman mapping, are shown.

We present a technique for Raman spectroscopic analysis of highly radioactive samples compatible with any standard micro-Raman spectrometer, without any radioactive contamination of the instrument. We also show some applications using actinide compounds and irradiated fuel materials.

一种新技术用于拉曼分析高放射性样品使用任何标准的微拉曼光谱仪

A novel approach for the Raman measurement of nuclear materials is reported in this paper. It consists of the enclosure of the radioactive sample in a ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

拉曼光谱和红外光谱电化学法作为分析共轭有机化合物的工具

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in human subjects. This information is very important for skin-related diseases and characterizing skin care product performance. This protocol illustrates a method for confocal Raman spectra collection and the subsequent analysis of the spectral dataset leveraging chemometrics. The goal of this method is to establish a standard protocol for data collection and provide general guidance for data analysis. Preprocessing (e.g., removal of outlier spectra) is a critical step when processing large datasets from clinical studies. As an example, we provide guidance based on prior knowledge of a dataset to identify the types of outliers and develop specific strategies to remove them. A principal component analysis is performed, and the loading spectra are compared with spectra from reference materials to select the number of components used in the final multivariate curve resolution (MCR) analysis. This approach is successful for extracting meaningful information from a large spectral dataset.

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

通过化学测量方法，从活体共聚焦拉曼光谱中解决水、蛋白质和脂质

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

电压在动态光散射粒子尺寸分析中的应用

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

超快时间解析近红外刺激拉曼测量功能 +偶联系统

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

A number of different procedures for preparing nanoparticles for surface analysis are presented (drop casting, spin coating, deposition from powders, and cryofixation). We discuss the challenges, opportunities, and possible applications of each method, particularly regarding the changes in the surface properties caused by the different preparation methods.

用于ToF-SIMS和XPS分析的纳米颗粒的制备

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, ...