基于Au@Carbon点纳米探针的无标记表面增强拉曼散射生物分析
Summary
在这项研究中，我们开发了一种低成本的基于表面增强拉曼散射（SERS）的指纹纳米探针，具有良好的生物相容性，以显示无标记活细胞生物成像并检测两种细菌菌株，详细展示了如何以无损方法获得活细胞的SERS光谱。
Abstract
表面增强拉曼散射（SERS）技术因其能够提供生物样品的分子指纹信息，以及在单细胞分析中的潜力，在生物医学领域受到越来越多的关注。这项工作旨在建立一种基于Au@carbon点纳米探针（Au@CDs）的无标记SERS生物分析的简单策略。在这里，多酚衍生的CD被用作还原剂，以快速合成核壳Au@CD纳米结构，由于协同拉曼增强机制，即使亚甲蓝（MB）的浓度低至10-9 M，也能提供强大的SERS性能。对于生物分析，Au@CDs可以作为独特的SERS纳米传感器来识别生物样品的细胞成分（例如癌细胞和细菌）。结合主成分分析，可以进一步区分不同物种的分子指纹图谱。此外，Au@CDs还可以进行无标记的SERS成像，以分析细胞内组成谱。该策略提供了一种可行的、无标记的SERS生物分析，为纳米诊断开辟了新的前景。
Introduction
单细胞分析对于揭示细胞异质性和评估细胞的综合状态至关重要。细胞对微环境的即时反应也保证了单细胞分析1。但是，当前技术存在一些限制。荧光检测可以应用于单细胞分析，但受到灵敏度低的限制。其他挑战来自细胞复杂的荧光背景和长期照射下的荧光光漂白2。表面增强拉曼散射（SERS）由于其优点，在单细胞分析方面可能符合条件，包括（1）反映固有的分子指纹信息和瞬时情况，（2）超高的表面灵敏度，（3）方便的多重检测，（4）高光稳定性，（5）检测可以量化进行比较分析，（6）避免近红外波长激发的细胞自发荧光，（7）可以在细胞水中进行检测环境，以及（8）检测可以指向小区3、4、5内的特定区域。
有两种广泛认可的机制可以将SERS理解为一种基本现象：电磁增强（EM）作为主要原因和化学增强（CM）。EM是指在给定频率的激发场中，当入射光的频率与金属中振荡的自由电子的频率相匹配时，由电磁波驱动的集体电子的振荡，从而产生表面等离子体共振（SPR）。当局部SPR（LSPR）通过入射激光撞击金属纳米颗粒（NPs）发生时，它会导致入射光的共振吸收或散射。因此，金属NPs的表面电磁场强度可以提高2到5个数量级4。然而，SERS大幅增强的关键不是单个金属NP，而是两个NP之间的差距，这会产生热点。CM从两个方面产生，包括（1）目标分子与金属NP之间的相互作用和（2）目标分子能够将电子转移到金属NP之间/从金属NP转移电子4,5。更详尽的细节可以在这些评论文章4,5中找到。以前的文献中已经提出了几种有前途的活细胞中SERS生物传感和成像方法，例如，检测凋亡细胞6，细胞器中的蛋白质7，细胞内miRNA，细胞内miRNA，细胞脂质膜，9细胞因子10和活细胞中的代谢物11，以及通过共聚焦SERS成像2鉴定和监测细胞，11,12,13.有趣的是，无标记SERS具有SERS的独特优势，可以描述内部分子光谱5。
无标记SERS的一个主要问题是合理可靠的底物。典型的SERS基板是贵金属NPs，因为它们具有出色的散射大量光的能力14。如今，纳米复合材料因其优异的物理化学性能和生物相容性而受到越来越多的关注。更重要的是，纳米复合材料可以表现出更好的SERS活性，因为纳米杂化物上的热点诱导了强烈的EM和来自其他非金属材料的额外化学增强15。例如，Fei等人使用MoS 2量子点（QD）作为还原剂来合成Au NP@MoS2QD纳米复合材料，用于小鼠4T1乳腺癌细胞（4T1细胞）的无标记近红外（NIR）SERS成像16。此外，Li等人制造了由Au NPs和2D二碲化铪纳米片组成的2D SERS底物，用于食源性致病菌的无标记SERS测量17。最近，良好的电子供体碳点（CDs）被用作还原剂，无需其他还原剂或辐照来合成Au@carbon点纳米探针（Au@CDs）18，据报道，基于金核和CD壳之间的电荷转移（CT）效应，碳点（CDs）是增强SERS活性的有效材料19,20。不仅如此，CD被认为是防止金NPs聚集的封端剂和稳定剂21。此外，它为与分析物的反应开辟了更多的可能性，因为它可以提供大量的结合和活性位点20。利用上述优势，Jin等人开发了一种快速可控的方法来制备具有独特SERS特性和出色催化活性的Ag@CD NPs，用于实时监测多相催化反应18。
本文展示了一种简单且低成本的方法来制造核壳Au@CD SERS底物，以鉴定细胞成分和无标记SERS活细胞生物成像，以及检测和区分 大肠杆菌 （大肠杆菌）和 金黄色葡萄球菌 （S. aureus），这有望为疾病的早期诊断和更好地了解细胞过程。
Protocol
1. Au@CDs的制造 注： 图 1 显示了Au@CDs的制造过程。 通过典型的水热处理程序18，使用柠檬酸（CA）和没食子酸（GA）制备CD溶液。将 100 μL 3.0 mg mL-1 制备的 CD 溶液加入 200 μL 10 mM 氯金酸 （HAuCl4）（参见材料表）中，在室温下持续 10 秒，直到产生紫色悬浮液。 在室温下以4,000…
Representative Results
Au@CDs的制造如图1所示。CD由CA和GA通过典型的水热工艺制备18。Au@CDs是在室温下通过CD在水性介质中还原HAuCl4来快速合成的。CD和Au@CDs的大小和形态可以通过TEM和高分辨率（HR）TEM23观察到。制备的CD是单分散的，尺寸接近2-6nm（图2A）。球形金核涂有一层约2.1nm的CD壳（图2B，C</s…
Discussion
综上所述，已经成功制造了具有2.1 nm超薄CD外壳的Au@CDs。纳米复合材料表现出比纯金NPs更高的SERS灵敏度。此外，Au@CDs在重现性和长期稳定性方面具有出色的性能。进一步的研究包括将Au@CDs作为底物，对A549细胞进行SERS成像31并检测两种细菌菌株32。已经证明，Au@CDs可以用作超灵敏的SERS探针，主要基于金NPs和CD之间的化学增强。
在以前的协议?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（32071399和62175071）、广州市科技计划（2019050001）、广东省基础与应用基础研究基金（2021A1515011988）和医学光电科学与技术教育部重点实验室（福建师范大学）开放基金（JYG2009）的支持。
Materials
| 10x PBS buffer (Cell culture) | Langeco Technology | BL316A | |
| 6 well cell culture plate | LABSELECT | 11110 | |
| Cell Counting Kit-8 (CCK-8) | GLPBIO | GK10001 | |
| Citric acid | Shanghai Aladdin Biochemical Technology | C108869 | |
| CO2 incubator | Thermo Fisher Technologies | 3111 | |
| Constant temperature magnetic agitator | Sartorius Scientific Instruments | SQP | |
| Cryogenic high speed centrifuge | Shanghai Boxun | SW-CJ-2FD | |
| DMEM high glucose cell culture medium | Procell | PM150210 | |
| Electronic balance | Sartorius Scientific Instruments | SQP | |
| Enzyme marker | Thermo Fisher Technologies | 3111 | |
| Fetal bovine serum | Zhejiang Tianhang Biological Technology | 11011-8611 | |
| Figure 1 | Figdraw. | ||
| Fourier infrared spectrometer | Thermo, America | Nicolet 380 | |
| Freeze dryer | Tecan | Infinite F50 | |
| Gallic acid | Shanghai Aladdin Biochemical Technology | G104228 | |
| Handheld Raman spectrometer | OCEANHOOD, Shanghai, China | Uspectral-PLUS | |
| HAuCl4 | Guangzhou Pharmaceutical Company (Guangzhou) | ||
| High resolution transmission electron microscope | Thermo Fisher Technologies | FEI Tecnai G2 Spirit T12 | |
| High temperature autoclave | Shanghai Boxun | YXQ-LS-50S  |
|
| Inverted microscope | Nanjing Jiangnan Yongxin Optical | XD-202 | |
| LB Broth BR | Huankai picoorganism | 028320 | |
| Medical ultra-low temperature refrigerator | Thermo Fisher Technologies | ULTS1368 | |
| Methylene blue | Sigma-Aldrich | ||
| Pancreatin Cell Digestive Solution | beyotime | C0207 | |
| Penicillin streptomycin double resistance | Shanghai Boxun | YXQ-LS-50S |
|
| Pure water meter | Millipore, USA | Milli-Q System | |
| Raman spectrometer | Renishaw | ||
| Sapphire chip | beyotime | ||
| Thermostatic water bath | Changzhou Noki | ||
| Ultra-clean table | Shanghai Boxun | SW-CJ-2FD | |
| Uv-visible light absorption spectrometer | MADAPA, China | UV-6100S | |
| Wire 3.4 | Renishaw |
References
- Zenobi, R. Single-cell metabolomics: analytical and biological perspectives. Science. 342 (6163), 1243259 (2013).
- Dong, C., et al. Simultaneous visualization of dual intercellular signal transductions via SERS imaging of membrane proteins dimerization on single cells. ACS Nano. 16 (9), 14055-14065 (2022).
- Lane, L. A., Qian, X., Nie, S. SERS nanoparticles in medicine: from label-free detection to spectroscopic tagging. Chemical Reviews. 115 (19), 10489-10529 (2015).
- Langer, J., et al. Present and future of surface-enhanced Raman scattering. ACS Nano. 14 (1), 28-117 (2020).
- Zong, C., et al. Surface-enhanced Raman spectroscopy for bioanalysis: reliability and challenges. Chemical Reviews. 118 (10), 4946-4980 (2018).
- Jiang, X., et al. Surface-enhanced Raman scattering-based sensing in vitro: facile and label-free detection of apoptotic cells at the single-cell level. Analytical Chemistry. 85 (5), 2809-2816 (2013).
- Qi, G., Diao, X., Hou, S., Kong, J., Jin, Y. Label-free SERS detection of protein damage in organelles under electrostimulation with 2D AuNPs-based nanomembranes as substrates. Analytical Chemistry. 94 (43), 14931-14937 (2022).
- Wang, J., et al. Trimer structures formed by target-triggered AuNPs self-assembly inducing electromagnetic hot spots for SERS-fluorescence dual-signal detection of intracellular miRNAs. Biosensors and Bioelectronics. 224, 115051 (2023).
- Živanović, V., Milewska, A., Leosson, K., Kneipp, J. Molecular structure and interactions of lipids in the outer membrane of living cells based on surface-enhanced Raman scattering and liposome models. Analytical Chemistry. 93 (29), 10106-10113 (2021).
- Cong, L., et al. Microfluidic droplet-SERS platform for single-cell cytokine analysis via a cell surface bioconjugation strategy. Analytical Chemistry. 94 (29), 10375-10383 (2022).
- Tan, Z., Zhu, C., Han, L., Liao, X., Wang, C. SERS and dark-field scattering dual-mode detection of intracellular hydrogen peroxide using biocompatible Au@ COF nanosensor. Sensors and Actuators B: Chemical. 373, 132770 (2022).
- Pan, X. T., et al. Super-long SERS active single silver nanowires for molecular imaging in 2D and 3D cell culture models. Biosensors. 12 (10), 875 (2022).
- Liu, Z., et al. A two-dimensional fingerprint nanoprobe based on black phosphorus for bio-SERS analysis and chemo-photothermal therapy. Nanoscale. 10 (39), 18795-18804 (2018).
- Bruzas, I., Lum, W., Gorunmez, Z., Sagle, L. Advances in surface-enhanced Raman spectroscopy (SERS) substrates for lipid and protein characterization: sensing and beyond. Analyst. 143 (17), 3990-4008 (2018).
- Li, D., et al. SERS analysis of carcinoma-associated fibroblasts in a tumor microenvironment based on targeted 2D nanosheets. Nanoscale. 12 (3), 2133-2141 (2020).
- Fei, X., et al. Synthesis of Au NP@MoS2quantum dots core@shell nanocomposites for SERS bio-analysis and label-free bio-imaging. Materials. 10 (6), 650 (2017).
- Li, Y., et al. Rapid label-free SERS detection of foodborne pathogenic bacteria based on hafnium ditelluride-Au nanocomposites. Journal of Innovative Optical Health Sciences. 13 (5), 2041004 (2020).
- Jin, J., et al. Precisely controllable core-shell Ag@ carbon dots nanoparticles: application to in situ super-sensitive monitoring of catalytic reactions. ACS Applied Materials & Interfaces. 8 (41), 27956-27965 (2016).
- Luo, P., Li, C., Shi, G. Synthesis of gold@ carbon dots composite nanoparticles for surface enhanced Raman scattering. Physical Chemistry Chemical Physics. 14 (20), 7360-7366 (2012).
- Li, L., et al. Accurate SERS monitoring of the plasmon mediated UV/visible/NIR photocatalytic and photothermal catalytic process involving Ag@carbon dots. Nanoscale. 13 (2), 1006-1015 (2021).
- Wang, X., et al. Reduced state carbon dots as both reductant and stabilizer for the synthesis of gold nanoparticles. Carbon. 64, 499-506 (2013).
- Zhu, M., et al. Physicochemical properties determine nanomaterial cellular uptake, transport, and fate. Accounts of Chemical Research. 46 (3), 622-631 (2013).
- Li, L., et al. SERS monitoring of photoinduced-enhanced oxidative stress amplifier on Au@ carbon dots for tumor catalytic therapy. Light: Science & Applications. 11 (1), 286 (2022).
- Fiori, F., et al. Highly photostable carbon dots from citric acid for bioimaging. Materials. 15 (7), 2395 (2022).
- Chen, X., et al. Preparation of carbon dots-based nanoparticles and their research of bioimaging and targeted antitumor therapy. Journal of Biomedical Materials Research. Part B, Applied Biomaterials. 110 (1), 220-228 (2022).
- Chen, M., et al. Red, green, and blue light-emitting carbon dots prepared from gallic acid for white light-emitting diode applications. Nanoscale Advances. 4 (1), 14-18 (2022).
- Byram, C., Moram, S. S. B., Shaik, A. K., Soma, V. R. Versatile gold based SERS substrates fabricated by ultrafast laser ablation for sensing picric acid and ammonium nitrate. Chemical Physics Letters. 685, 103-107 (2017).
- Efrima, S., et al. Understanding SERS of bacteria. Journal of Raman Spectroscopy. 40 (3), 277-288 (2009).
- Movasaghi, Z., Rehman, S., Rehman, I. U. Raman spectroscopy of biological tissues. Applied Spectroscopy Reviews. 42 (5), 493-541 (2007).
- Mushtaq, A., et al. Surface-enhanced Raman spectroscopy (SERS) for monitoring colistin-resistant and susceptible E. coli strains. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 278, 121315 (2022).
- Mosier-Boss, P. A., Sorensen, K. C., George, R. D., Obraztsova, A. SERS substrates fabricated using ceramic filters for the detection of bacteria. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 153, 591-598 (2016).
- Zhang, P., et al. Dynamic insights into increasing antibiotic resistance in Staphylococcus aureus by label-free SERS using a portable Raman spectrometer. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 273, 121070 (2022).
- Li, J. F., Zhang, Y. J., Ding, S. Y., Panneerselvam, R., Tian, Z. Q. Core-shell nanoparticle-enhanced Raman spectroscopy. Chemical Reviews. 117 (7), 5002-5069 (2017).
- Bodelon, G., Montes-Garcia, V., Perez-Juste, J., Pastoriza-Santos, I. Surface-enhanced Raman scattering spectroscopy for label-free analysis of P. aeruginosa quorum sensing. Frontiers in Cellular and Infection Microbiology. 8, 143 (2018).
- Weiss, R., et al. Surface-enhanced Raman spectroscopy of microorganisms: limitations and applicability on the single-cell level. Analyst. 144 (3), 943-953 (2019).
- Oliveira, K., et al. Multiplex SERS phenotyping of single cancer cells in microdroplets. Advanced Optical Materials. 11 (1), 2201500 (2023).
- Ho, C. S., et al. Rapid identification of pathogenic bacteria using Raman spectroscopy and deep learning. Nature Communications. 10 (1), 4927 (2019).
- Spedalieri, C., Kneipp, J. Surface enhanced Raman scattering for probing cellular biochemistry. Nanoscale. 14 (14), 5314-5328 (2022).
- Weng, S. Y., et al. Highly sensitive and reliable detection of microRNA for clinically disease surveillance using SERS biosensor integrated with catalytic hairpin assembly amplification technology. Biosensors & Bioelectronics. 208, 114236 (2022).
- Wang, J. W., et al. Target-triggered nanomaterial self-assembly induced electromagnetic hot-Spot Generation for SERS-fluorescence dual-mode in situ monitoring MiRNA-guided phototherapy. Analytical Chemistry. 93 (41), 13755-13764 (2021).
Cite This Article
Zheng, Y., Xiao, X., Li, Z., Shao, Y., Chen, J., Guo, Z., Zhong, H., Liu, Z. Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes. J. Vis. Exp. (196), e65524, doi:10.3791/65524 (2023).