Single-molecule surface-enhanced Raman spectroscopy (SM-SERS): characteristics and analysis

Zhao Xing; Hao Qi; Ni Zhen-Hua; Qiu Teng

doi:10.7498/aps.70.20201447

Abstract

Single-molecule detection (SMD), which represents the detection limit in molecular spectroscopy, has opened a new research realm in the fields of catalysis, DNA sequencing and protein analysis. Meanwhile, it provides new insights into the understanding of the molecule behaviors in a complex system. Specifically, SMD enables the quantitatively identifying of molecules accurate to single digit, provides the molecular distribution state under specific environments, and permits the in-situ observation of signal fluctuations of a single-molecule under chemical stimulus. Single-molecule surface-enhanced Raman spectroscopy (SM-SERS) is a new subject in SMD which features specific recognition of molecules by identifying the molecular chemical bonds. It is a non-destructive technology which reflects the vibration energy and rotational energy information of molecules. This technique employs metallic nanostructures to form surface plasmon resonances (SRP) under external excitation. The SPRs generate strong local electromagnetic fields ("hot spots") around metal surface to amplify the Raman signal of probe molecules in the vicinity of plasmonic materials. The giant field enhancement endows SERS superior sensitivity in trace molecule detection down to a single-molecule level. The SM-SERS offers a facile method to track the evolution of a single molecule, revealing the reaction pathways, adsorption state and distributions, and charge exchanges between the molecule and surrounding environment. Though SM-SERS has been proposed more than 20 years ago, the acquisition of SM-SERS spectra remains a bottleneck in this field due to the disability in judging the origins of these spectra. On the other hand, the lack of knowledge in analyzing SM-SERS spectra also limits the development of SM-SERS as the origins of molecule behavior at a micro level is basically unknown to the public. This review paper covers the development of SM-SERS, the past and current methods of verifying SM-SERS including the non-statistical and the bi-analyte statistical methods, the investigation into the understanding of the fluctuation characteristics of SM-SERS, as well as the related mechanisms with regard to the unique phenomena in SM-SERS such as molecule diffusion, spectral blinking and broadening. We hope this review can help the readers to relate the characteristics in SM-SERS with the origins of molecular variations during the detection, in this way to get a clear and in-depth understanding of the roadmap for SM-SERS.

Keywords:

Authors and contacts

1.
School of Physics, Southeast University, Nanjing 211189, China
2.
Quantum Information Research Center, Southeast University, Nanjing 211189, China

Corresponding author: Hao Qi, qihao@seu.edu.cn

Funds: Project supported by the National Natural Science Foundation of China (Grant No. 22004016) and the Fundamental Research Funds for the Central Universities, China (Grant Nos. 2242021R41069, 2242021R10100).

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Cited By

图 1 SM-SERS与普通SERS光谱图的区别^[21]

Figure 1. Typical spectra of SM-SERS and traditional SERS^[21].

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图 2 (a) DNA在修饰的金@银纳米粒子壳层结构之间构造“间隙”结构用于SM-SERS检测^[30]; (b)扫描隧道显微镜控制的TERS的示意图, 其中V_b是偏置电压, I_t是隧道电流^[33]; (c)高分辨靶分子TERS光谱与图像^[33]; (d)相邻分子间的TERS成像(上)及沿图中轨迹线进行十次连续TERS测量的相应光谱演化(下)^[34]; (e)分子亚纳米分辨率STM图像(左)以及单个分子偶极矩模拟计算图(右)^[36]; (f) DNA单链片段的TERS图像^[38]

Figure 2. (a) The Gap structure constructed by DNA modified gold@silver nanoparticles for SM SERS detection^[30]; (b) schematic diagram of TERS controlled by scanning tunneling microscope, where V_b is the sampling bias voltage and I_t is the tunnel current^[33]; (c) high-resolution TERS spectrums and images of target molecules^[33]; (d) TERS imaging between adjacent molecules (up) and the corresponding spectral evolution of ten consecutive TERS measurements along the trajectory in the left image (down)^[34]; (e) molecular sub-nanometer resolution STM image (left) and single molecule dipole moment simulation calculation diagram (right)^[36]; (f) TERS image of DNA single-stranded fragments^[38].

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图 3 不同散射截面分子实现SM-SERS检测所要满足的电磁增强EF^[43]

Figure 3. Electromagnetic enhancement EF value of molecules with different cross sections required for SM-SERS detection^[43].

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图 4 SM-SERS技术用于监测原位催化反应过程　(a) 单个4-NTP分子通过原位催化反应生成硫酚分子的SERS光谱图^[44]; (b)单分子罗丹明B异硫氰酸酯经历不同光照时间催化生成不同物质所对应的光谱图^[45]

Figure 4. Monitoring in situ catalytic reaction via SM-SERS: (a) SERS spectra of Thiophenol generated by single-molecule 4-NTP via in situ catalytic reaction^[44]; (b) SERS Spectra of Rhodamine B isothiocyanate catalyzed by single-molecule rhodamine B under different illumination time^[45].

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图 5 双分析物测量法获取SM-SERS　(a) Rh6G事件、CV事件和混合事件的SERS光谱图(左)及三种事件的统计直方图(右)^[49]; (b)不同同位素标记Rh6G后获得的三种不同独立事件SERS光谱(左)及三种事件发生次数统计图(右边)^[51]

Figure 5. Typical SM-SERS spectra in bi-analyte measurements: (a) SERS spectra of an Rh6G event, CV event and mixed event (left), and statistical histograms of the three events (right)^[49]; (b) SERS spectra of three different independent events (left), and statistics of the three events (right) by labelling Rh6G with different isotopes^[51].

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图 6 单个分子在热点区域内发生漂移示意图, 其中E_λ是入射光的电场, $ {{K}}' $表示波矢, O是“热点”中心

Figure 6. The schematic diagram of the diffusion of a single-molecule in the hot spot area. Where E_λ is the electric field of the incident light, $ {{K}}' $ is the wave vector and O is the "hot spots" center.

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图 7 单个分子漂移行为的探究^[58]　(a) SERS质心位置在二维空间中出现的次数(右边色条代表事件次数); (b) 在每个质心位置测得的平均SERS强度, 其中纳米粒子发射质心设置为(0, 0); (c) SRER质心的位置和强度随时间发生变化, 这表明单个探针分子在热点附近发生漂移

Figure 7. Study on the diffusion behavior of a single molecule^[58]: (a) The number the SERS centroid position that appears in two-dimensional space during the detection (the right color bar represents the number of events); (b) the average SERS intensity measured at each centroid, where the emission centroid of nanoparticles is set to (0, 0); (c) the intensity and position of SERS centroid change with time, which indicates that single molecule wanders around the hot spots.

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图 8 SM-SERS中分子不同取向对光谱产生波动影响^[72]　(a) 银纳米腔体结构示意图; (b) 光谱1—3表示纳米间隙中不同取向分子的SM-SERS信号, 光谱4表示高浓度普通SERS光谱; (c) Rh6G分子的结构以及不同场方向在分子α-γ和α-β平面上的投影

Figure 8. The effects of molecular orientations on spectrum fluctuations in SM-SERS^[72]: (a) Schematic diagram of silver nanocavity structure; (b) spectrum 1–3 is SM-SERS signals of molecules with different orientations in the nan-gap, and spectrum 4 is the SERS spectrum of high concentration molecules; (c) the chemical structure of Rh6G molecule and the projection of different field directions on the α-γ and α-β planes of this molecule.

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图 9 (a)“银纳米球-金膜”纳米腔体结构用于SERS测量及纳米腔暗场散射光谱^[65]; (b) 催化反应生成中间产物的间歇性光谱特征轨迹^[65]; (c) 在开(红)和关(蓝)期间从图9(b)中取样的SERS光谱, 两者光谱的差异(橙)及其协方差矩阵V_X = 1364 cm^–1分量(绿). 在1160, 1314, 1364 cm^–1处出现间歇性SERS峰归因于中间产物羟胺基苯硫醇分子的拉曼峰^[65]; (d)光谱的上升沿阶跃频率f_up(左)和下降沿阶跃频率f_down(右)与4-NTP分子的衰减速率常数k_NBT之间的关系^[65]

Figure 9. (a) The “silver nanosphere-gold film” nanocavity structure in SERS measurement^[65] (This figure contains chemical structures of NBT and DMAB molecules, as well as the dark field scattering spectrum of a single nanostructure cavity); (b) the trace of the intermittent spectral characteristics of intermediates generated by catalytic reaction^[65]; (c) SERS spectra sampled from Fig. 9(b) during on (red) and off (blue), the differences between the two spectra (orange) and the V_X = 1364 cm^–1 component (green) of the covariance matrix. The intermittent SERS peaks at 1160, 1314 and 1364 cm^–1 are attributed to the Raman peaks of the intermediate product hydroxyl aminobenzenethiol^[65]; (d) the relationship between the spectral step-up frequency f_up (left) and step-down frequency f_down (right) and the decay rate constant k_NBT of 4-NTP molecule^[65].

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图 10 SM-SERS的闪烁现象^[78]: 单个Rh6G分子SERS信号随时间变化的轨迹图, 其中在波数614 cm^–1和774 cm^–1处发生明显的光谱波动

Figure 10. The Blinking phenomenon in SM-SERS ^[78]: The time-dependent trajectories of SERS signal of a single Rh6G molecule. Obvious spectral fluctuations are observed at 614 cm^–1 and 774 cm^–1.

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图 11 高速SERS信号采集和超分辨率成像^[79]　(a) SM-SERS放大后的两个波动图, 每个持续约0.3 ms, 相距61 ms; (b)光电探测器上接收到两个易于区分的光谱波动事件成像; (c)在纳米粒子的空间位置上绘制的三个光谱波动轨迹

Figure 11. High-speed SERS acquisition and super-resolution SERS imaging^[79]: (a) Each of two amplified wave patterns of SM-SER spectra lasted for about 0.3 ms and separated by 61 ms; (b) images of spectral fluctuation events; (a) three spectral wave trajectories on the spatial position of nanoparticles.

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图 12 SM-SERS光谱的展宽现象　(a)单个尼罗蓝染料分子在590 cm^–1能量振动处的不均匀展宽现象^[62]; (b)单个罗丹明800分子在2226 cm^–1能量振动处的不均匀展宽现象^[62]; (c) SM-SERS事件中单个尼罗蓝分子在590 cm^–1处光谱的半高宽(Γ)与温度(T)之间的关系, 表明随着温度T升高SM-SERS光谱发生均匀展宽^[21]

Figure 12. Broadening of SM-SERS spectrum: (a) Inhomogeneous broadening of a single Nile Blue dye molecule at 590 cm^–1 energy vibration^[62]; (b) inhomogeneous broadening of a single Rhodamine 800 molecule at 2226 cm^–1 energy vibration^[62]; (c) the relationship between the FWHM (Γ) of a single Nile Blue molecule indifferent SM-SERS events at 590 cm^–1 with increasing temperature T. The result indicates that the SM-SERS spectrum broadens uniformly with the increase of temperature (T)^[21].

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