柔性表面增强拉曼光谱芯片制备 
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Objective Surface-enhanced Raman spectroscopy (SERS) can provide fingerprint information on target molecules with high detection sensitivity without being affected by water, which makes it an attractive non-destructive analysis technology. The SERS active substrate is key part of inspection applications. Therefore, a significant research effort has been devoted to the development of a stable, uniform, and repeatable SERS substrate. Unlike rigid SERS substrate, flexible SERS substrate can be flexibly deformed, which is convenient for in situ detection on irregular curved surfaces and can even be used for wipe sampling detection directly. However, the characteristics of the flexible substrate itself significantly impact the SERS performance, and the commonly used sol drop casting method to prepare SERS active substrates is significantly affected by the “coffee ring” effect. Because the effect caused uneven distribution of nanoparticles, uniformity of the SERS signal is affected. In this paper, simulation analysis and experimental research on the reflectivity of the substrate are carried out, and optimization of the substrate is performed to suppress the “coffee ring” effect. A high-throughput and array-type flexible SERS chip with excellent performance is successfully fabricated, which has application potential in biomedicine, food safety, environmental pollution, and other detection fields.
Methods To study the influence of substrate reflectivity on SERS performance, the Raman signal intensities of different substrates are compared and analyzed by simulation (COMSOL Multiphysics) and experimental tests. To eliminate the “coffee ring” effect generated from the evaporation of nanoparticle suspensions, we control the solvent composition by adding a certain proportion of ethylene glycol in Ag sol, and use inward Marangoni flow induced by the surface tension gradient, which ensure uniform deposition of nanoparticles. The array detection unit of the SERS chip is fabricated on aluminum foil through laser printing. Then, Ag sol mixed liquid is dropped into the detection area and confined by the hydrophobic toner film. The SERS chip is formed after the droplets dried in a vacuum drying oven. Rhodamine 6G (R6G) is used as a probe molecule for the SERS test, and the performance of the SERS chip is evaluated by calculating the Raman enhancement factor and performing a signal uniformity test.
Results and Discussions Simulation analysis results show that the greater the reflectivity of the substrate, the higher the intensity of the Raman signal. The Raman detection results for R6G molecules also show that the substrate with high reflectivity is helpful for Raman signal collection, which is consistent with the simulation results. Aluminum foil is chosen as the substrate because it has the strongest reflectivity under 532nm excitation light among the several materials used, and the SERS chip is manufactured using the Ag sol drop casting method. After adding ethylene glycol to the Ag sol, a tension gradient is formed on the surface of the droplet, resulting in an inward Marangoni flow, which prevents the nanoparticles from gathering on the edge of the droplet, thereby suppressing the “coffee ring” effect. The experimental results show that when 200μL of ethylene glycol is added to 1mL of Ag sol, the “coffee ring” effect is eliminated, and the Ag nanoparticles are uniformly distributed on the substrate. The Raman test results indicate that the SERS chip exhibited a high Raman enhancement factor of up to 1.32×10 8 and a detection limit of down to 10 -11 mol for R6G molecules. Furthermore, the chip showes good signal uniformity.
Conclusions Flexible SERS chips have many advantages in Raman detection applications. In this work, simulation analysis and experimental tests show that the high reflectivity of the substrate has a significant impact on improving the SERS performance of the chip. By adding a certain proportion of ethylene glycol solution to the Ag sol, the surface tension state of the solution can be changed, ensuring uniform distribution of silver nanoparticles on the chip, thereby eliminating the “coffee ring” effect generated during deposition of the nanoparticles. The flexible SERS chip shows good Raman detection performance. The array structure of the SERS chip enables it to achieve high-throughput, multiparameter detection. It has strong application potential in fields such as biomedicine, food safety, and environmental pollution.
1 引言
表面增强拉曼光谱(SERS)可以提供目标分子的指纹信息,其是一种极具吸引力的无损分析技术[1-3],具有检测灵敏度高以及不受水干扰的优点,在生物医学、食品安全和环境污染等领域具有极大的应用潜力[4-9]。在SERS技术的应用过程中,选择和制备合适的SERS活性基底成为了其中的关键一环,因此大量的研究都致力于开发稳定性高、均匀性佳和重复性好的SERS基底[10-12]。
等离子体纳米结构阵列是SERS基底的一种主要构造形式。使用先进的微/纳米加工技术,如电子束光刻(EBL)[13]、纳米压印光刻(NIL)[14]和聚焦离子束(FIB)[15]等,可以制备高度均匀的纳米阵列SERS基底,但是相对较高的成本和复杂的操作程序限制了其应用范围,而贵金属纳米颗粒是产生密集SERS“热点”的最经济且最方便的材料之一。采用成熟的化学方法可以批量地合成各种形貌和尺寸的纳米材料,如纳米星、纳米球和纳米线等[16-18]。采用滴铸法可以直接将贵金属纳米颗粒组装在衬底上并进行SERS检测,该方法是一种简单、经济且常用的方法[19-20]。然而,从蒸发纳米颗粒悬浮液到纳米颗粒组装的过程中不可避免地会产生“咖啡环”。补偿液滴与衬底的接触线处因溶剂损失而引起纳米颗粒沿径向扩散,使得大量的纳米颗粒被扩散到液滴的边沿,这将导致纳米颗粒分布得不均匀[21-22]。纳米颗粒分布不均会导致“热点”不稳定,进而影响拉曼信号的重复性和可靠性。为了消除咖啡环的影响并保证拉曼信号的整体均匀性和灵敏度,科研人员对“热点”的均匀性进行了广泛的研究[23-27]。根据超疏水表面对液滴有弱或无钉扎力的特点来消除咖啡环以增加SERS强度[23-26],但是制备过程通常很复杂,其中始终需要理想的表面粗糙度、层次结构或精确的化学组成才能实现较好的富集结果。Yang等[25]提出了一种具有优异SERS性能的超疏水SERS基底,制备过程中需要采用具有纳米级孔径的特氟龙膜并使用全氟化液体渗透技术,制备过程复杂并增加了成本。
通常,大多数SERS基底均是采用硅片和玻璃等硬质衬底来制备的。刚性SERS基底仅可以在平面上原位检测分析物,缺乏灵活性,实际应用中具有很大的局限性。近年来,柔性SERS基底吸引了科研人员的大量关注,多种柔性材料被用作衬底以制备SERS基底[28-31]。与刚性材料不同,柔性SERS基底能够灵活多变,便于不规则曲面上的原位检测,甚至可以直接用于擦拭取样的检测[32-33]。各种柔性材料中,纸张具有廉价、环保和易回收等独特的优势。目前,滤纸、打印纸和色谱纸等都被用来制备柔性SERS基底[34-38],但是纸张表面存在许多微米级且不规则的孔状缺陷,这些缺陷一方面会导致入射激光能量散射损失,从而降低电磁耦合的总强度和拉曼信号强度[37],另一方面纸张表面的高粗糙度可能会大于拉曼系统,从而造成实际检测的拉曼信号强度相对较弱。同时,某些纸张有强烈的荧光信号,这会进一步对拉曼信号造成干扰。在简单的制备工艺和低成本的要求下,制备具有高SERS活性、均匀“热点”和良好重现性的SERS基底仍然面临巨大的挑战。
本文提出一种新颖、简单且灵活的方法制备铝箔纸SERS基底,该方法使用激光碳粉打印机制作阵列式检测区单元,通过控制溶剂的组成来抑制咖啡环的产生,这可以使纳米颗粒分布更均匀,从而提高热点的均匀性和SERS性能。铝箔纸具有纸张柔性的特点,因此不会被液体浸润,而且不会出现褶皱的现象。最重要的是,铝在可见光到红外范围内具有很高的反射率,有利于对拉曼信号的收集。当进行SERS检测时,样品分子发出的拉曼信号会向四面八方散射,而铝衬底会将一部分射向衬底的拉曼信号反射到物镜,从而提高SERS信号的强度。
2 实验与仿真
2.1 实验材料
硝酸银(AgNO3, 分析纯)、柠檬酸三钠(分析纯)、浓硫酸(H2SO4,质量浓度为98%)和过氧化氢(H2O2,质量浓度为30%)购自重庆川东化工(集团)有限公司。罗丹明6G(R6G,分析纯)和乙二醇(EG,质量浓度为99.9%)购自重庆西南化学试剂有限公司。<100>晶向硅片购自苏州晶矽电子科技有限公司。实验使用的玻璃器皿均先使用食人鱼(Piranha)溶液洗涤,再使用去离子水对洗涤后的玻璃器皿进行彻底冲洗并在使用前干燥,其中去离子水的电阻率为18.2MΩ·cm。
2.2 银溶胶制备
采用柠檬酸三钠还原硝酸银的方法制备银溶胶[17],具体步骤如下。首先将100mL的浓度为1mmol/L的AgNO3水溶液置于磁力搅拌加热器中并加热至沸腾;然后在剧烈搅拌下迅速加入2mL的质量分数为1%的柠檬酸钠溶液,继续搅拌并加热约20min,待溶液颜色变成黄绿色,表明银溶胶制备成功。将制备的银溶胶冷却至室温,通过孔径为0.22μm的Millipore膜过滤后置于棕色瓶中并在4 ℃的温度下储存备用。使用前,对银溶胶使用去离子水进行离心清洗三次。
2.3 衬底反射率对SERS的影响
利用COMSOL Multiphysics软件中的射线光学模块进行仿真,研究不同反射率的衬底对拉曼信号强度的影响。几何模型包含一个代表衬底的“物面”和一个代表拉曼信号收集物镜的“镜面”。假设拉曼信号沿着球面的各个方向均匀散射,且不同方向的拉曼信号具有相同的功率,拉曼信号到达“镜面”后被物镜“收集”。通过计算“镜面”的入射热通量来表示物镜收集的拉曼信号强度,进而比较不同反射率的衬底对拉曼信号强度的影响。将具有相同表面粗糙度的金、铝、镍和硅作为衬底,将同一浓度的罗丹明6G溶液直接滴在不同反射率的衬底上,并在相同的条件下收集拉曼信号,通过比较拉曼目标分子的拉曼信号强度来分析衬底对拉曼信号的影响。
2.4 SERS芯片的制备
实验选取4组银溶胶,每组均取1mL,分别加入0,100,200,500μL乙二醇溶液并使用超声波处理2 min,使其充分混合均匀。使用移液枪各取6μL混合液并滴在衬底上,将其放入真空加热箱中进行加热,直至溶液完全干燥。制备SERS芯片的方法如下:1)将铝箔粘贴在A4纸上,保持表面平整无褶皱;2)使用激光碳粉打印机在铝箔表面打印阵列式圆形检测区单元,圆形检测区的直径约为2mm;3)使用移液枪将银溶胶滴在圆形检测区内,每次滴加6μL,将其置于真空干燥箱中恒温加热至干燥,直至液滴完全干噪。重复上述步骤三次,使得银纳米颗粒均匀地分布在检测区内。
2.5 实验仪器
使用UV-2450分光光度计来测量银溶胶的紫外-可见光谱,记录的波长范围为300~900nm。银纳米颗粒的表面形貌使用TESCAN公司生产的场发射扫描电镜(FESEM)来表征。使用激光共聚焦拉曼光谱仪来收集拉曼光谱,激光器的输出波长为532nm。真空干燥箱购自河北豪威电器设备科技有限公司。
3 分析与讨论
3.1 银纳米颗粒的光学特性
银纳米颗粒具有很好的拉曼增强性能,常用于制备SERS基底。银溶胶的紫外-可见-红外吸收光谱,如
图 1. 银纳米颗粒的表征结果。(a)紫外-可见光谱,插图为FESEM图像;(b)银纳米颗粒的直径分布情况
Fig. 1. Characterization results of Ag nanoparticles. (a) Ultraviolet-visible spectrum, inset is FESEM image; (b) diameter distribution of Ag nanoparticles
3.2 衬底的反射作用对拉曼信号的影响
由拉曼散射的原理可知,当激发光照射在分析物的分子上时,分子发出的拉曼信号会随机向各个方向散射,而只有进入物镜孔径角内的拉曼信号才会被收集。对于固体SERS基底,一般采用背向方式来收集拉曼信号。根据光学原理可以推断衬底的反射作用会增大进入物镜孔径角内的拉曼信号强度。
为了定量分析衬底的反射作用对增强拉曼信号的贡献,使用COMSOL Multiphysics软件中的射线光学模块进行有限元模拟。当衬底发生反射时,收集拉曼信号的原理如
图 2. 衬底对拉曼信号的影响。(a)收集拉曼光的原理示意图;(b)不同反射率的光能分布;(c)不同反射率的光能密度曲线;(d)不同衬底的相对反射率;(e)在拉曼位移为1360cm-1处不同衬底的峰值;(f)仿真结果和实验结果的归一化曲线
Fig. 2. Influence of substrates on Raman signals. (a) Principle diagram of collecting Raman light; (b) light energy distribution with different reflectivity; (c) light energy density curves with different reflectivity; (d) relative reflectivity of different substrates; (e) peak values of different substrates at Raman displacement is 1360cm-1; (f) normalized curves of simulation result and experimental result
3.3 SERS芯片的性能
滴在衬底上的银溶胶在蒸发干燥的过程中,咖啡环会使基底上的银纳米颗粒不能均匀分布。在1mL的银溶胶中混合0,100,200,500μL的乙二醇溶液,干燥后的图片如
图 3. SERS芯片的制备。(a)含有不同比例乙二醇的银溶胶干燥后的显微图像;(b)阵列式检测区及银溶胶液滴图片; (c)检测区中银纳米颗粒的分布情况
Fig. 3. Preparation of SERS chip. (a) Microscopic images of Ag sol containing different proportions of ethylene glycol after drying; (b) picture of array detection area and silver sol droplets; (c) distribution of Ag nanoparticles in detection area
为了消除咖啡环,在银溶胶中混入不同比例的乙二醇溶液以改变溶液的组成成分,使混合溶液的液滴表面形成张力梯度以抑制咖啡环,从而解决银纳米颗粒不能均匀分布的问题。为了缩短混合溶剂的挥发时间,同时避免加热过程中银纳米颗粒和铝箔衬底被严重氧化,则使用真空干燥箱对其进行加热以蒸发溶剂,其中真空干燥箱的真空度约为130 Pa,温度为70 ℃。从
虽然在银溶胶中混合乙二醇可以使基底上的银纳米颗粒均匀分布,但是从实验结果可以看到,对于相同的操作方式,银纳米颗粒在衬底上形成的斑点形状和大小各异,这在一定程度上影响SERS基底的可重复性。根据文献[
42]的研究结果可知,使用碳粉作为疏水屏障以制作检测区,将银溶胶液滴限制在检测区的内部,可以使银纳米颗粒的沉积面积固定,从而保证SERS基底的可重复性。同时,当进行SERS检测时,疏水屏障可以将分析物溶液限制在检测区的内部,避免其扩散到周围环境,从而将各个检测区域彼此隔离。从
3.4 SERS性能测试
将R6G作为探针分子,用来评估SERS芯片发出拉曼信号强度的增强性能。为了确保实验结果更可靠,在检测区上随机选择10个点的SERS光谱并求平均值后作为最终结果。
图 4. SERS芯片的性能测试结果。(a)不同浓度R6G溶液的SERS;(b)SERS芯片的SERS映射图;(c)弯折的SERS芯片;(d)芯片弯折多次后的SERS映射图
Fig. 4. SERS chip performance test results. (a) SERS of different concentrations of R6G solution; (b) SERS mapping of SERS chip; (c) bending SERS chip; (d) SERS mapping of chip after bending for many times
为了评估SERS芯片的SERS活性,将R6G溶液的SERS光谱中在拉曼位移为1360cm-1处的峰强度与其无增强的对应峰强度进行比较,用来计算拉曼增强因子(EF)。增强因子的计算公式为xEF=
实际应用中,信号的均匀性是评估SERS芯片性能的最重要参数之一,实验中利用SERS映射对芯片的信号进行均匀性表征。在检测区域上取50μm×50μm并沿X轴和Y轴均以2μm的步长进行扫描,激光功率和积分时间分别为0.125mW和1s,共收集625个拉曼光谱。以R6G溶液在1360cm-1处的特征峰强度绘制SERS映射图,结果如
弯折的SERS芯片如
4 结论
拉曼检测应用中,柔性的SERS芯片具有诸多优势。将高反射率的材料作为衬底,采用最简单的滴铸法来制备柔性的SERS芯片。对不同反射率的衬底进行仿真分析和实验测试,可以看到衬底的反射率对拉曼信号的收集有极大的影响。通过对比几种材料,铝箔在波长为532nm激发光具有很高的反射率,因此选择铝箔作为衬底。为了解决从蒸发纳米颗粒悬浮液到纳米颗粒组装过程中存在“咖啡环”的问题,通过在银溶胶中添加一定比例的乙二醇来改变溶剂成分,使液滴表面形成张力梯度。利用表面张力梯度引起向内Marangoni流动以抑制咖啡环的产生,使芯片上的银纳米颗粒更能均匀分布。最后以R6G溶液为探针分子,对SERS芯片进行拉曼测试。实验结果表明,柔性SERS芯片的增强因子高达1.32×108,对R6G溶液的检测限低至10-11mol/L,芯片可以表现出良好的信号均匀性,多次弯折的SERS芯片并不会影响拉曼信号的检测。SERS芯片的阵列式结构能够实现高通量且多参数的检测,在生物医学、食品安全和环境污染等检测领域具有很大的应用潜力。
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Article Outline
杨峰, 文枰, 张志强, 李丹阳, 陈李, 李顺波, 徐溢. 柔性表面增强拉曼光谱芯片制备[J]. 中国激光, 2021, 48(1): 0113001. Feng Yang, Ping Wen, Zhiqiang Zhang, Danyang Li, Li Chen, Shunbo Li, Yi Xu. Fabrication of Flexible Surface-Enhanced Raman Spectroscopy Chip[J]. Chinese Journal of Lasers, 2021, 48(1): 0113001.
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