Name: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging (Video) | JoVE
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This work was supported by the National Natural Science Foundation of China (32071399 and 62175071), the Science and Technology Program of Guangzhou (2019050001), the Guangdong Basic and Applied Basic Research Foundation (2021A1515011988), and the Open Foundation of the Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, China (JYG2009).

1. Fabrication of Au@CDs
NOTE: Figure 1 illustrates a fabrication procedure for Au@CDs.
<ol>
	<li>Prepare CD solution using citric acid (CA) and gallic acid (GA) via a typical hydrothermal treatment procedure18. Add 100 &#181;L of 3.0 mg mL-1 of the prepared CD solution into 200 &#181;L of 10 mM chloroauric acid (HAuCl4) (see Table of Materials) at room temperature for 10 s ...

Label-Free SERS Analysis: Au@Carbon Dot Nanoprobes for Molecular Fingerprinting

<ol>
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In summary, Au@CDs with an ultrathin CD shell of 2.1 nm have been successfully fabricated. The nanocomposites show superior SERS sensitivity than pure Au NPs. Also, Au@CDs possess excellent performance in reproducibility and long-term stability. Further research includes taking Au@CDs as substrates to perform SERS imaging of A549 cells31&#160;and to detect two bacterial strains32. It has been proved that Au@CDs can be used as an ultrasensitive SERS probe mainly based on the...

Single-cell analysis is essential for the study of revealing cellular heterogeneity and assessing the comprehensive state of the cell. The cell&#39;s instant response to the microenvironment also warrants single-cell analysis1. However, there are some limitations to the current techniques. Fluorescence detection can be applied to single-cell analysis, but it&#39;s limited by low sensitivity. Other challenges arise from the complicated fluorescence background of cells and the fluorescence photobleaching under long-term irradiation2. Surface-enhanced Raman scattering (SERS) may qualify in terms of single-cell analysis owing to its advantages, including (1) reflecting the intrinsic molecular fingerprint information and the instantaneous situation, (2) ultrahigh surface sensitivity, (3) convenient multiplex detection, (4) high photostability, (5) detection can be quantified for comparative analysis, (6) avoiding cellular autofluorescence with the NIR wavelength excitation, (7) detection can be performed in a cellular aqueous environment, and (8) detection can be directed to a specific region within the cell3,4,5.
There are two broadly recognized mechanisms to understand SERS as a fundamental phenomenon: electromagnetic enhancement (EM) as a dominant reason and chemical enhancement (CM). EM refers to, in a given frequency of the exciting field, the oscillation of collective electrons driven by electromagnetic waves when the frequency of the incident light matches the frequency of free electrons oscillating in the metal, giving rise to surface plasmon resonance (SPR). When localized SPR (LSPR) occurs through the incident laser impinging at the metal nanoparticles (NPs), it leads to the resonant absorption or scattering of the incident light.&#160;Consequently, the surface electromagnetic field intensity of metal NPs can be enhanced by two to five orders4. However, the key to the huge enhancement in SERS is not a single metal NP, but the gap between two NPs, which creates hot spots. CM is generated from two sides, including (1) interactions between target molecules and metal NPs and (2) target molecules being able to transfer electrons to/from metal NPs4,5. More exhaustive details can be found in these review articles4,5. Several promising methods for SERS biosensing and imaging in living cells have been presented in previous literature, for example, the detection of apoptotic cells6, proteins in organelles7, intracellular miRNAs8, cellular lipid membranes,9cytokines10, and metabolites11&#160;in living cells, as well as the identification and monitoring of cells by confocal SERS imaging2,11,12,13. Interestingly, label-free SERS presents the unique advantage of SERS, which can describe internal molecular spectra5.
A major issue for label-free SERS is a rational and reliable substrate. Typical SERS substrates are noble metal NPs because of their excellent capacity to scatter a lot of light14. Nowadays, more and more attention is paid to nanocomposites due to their remarkable physical and chemical properties and biocompatibility. More significantly, nanocomposites can show better SERS activity because of the intense EM induced by the hot spots on the nanohybrids and additional chemical enhancement originating from other non-metal materials15. For example, Fei et al. used MoS2quantum dots (QDs) as reducers to synthesize Au NP@MoS2 QD nanocomposites for label-free near-infrared (NIR) SERS imaging of mouse 4T1 breast cancer cell (4T1 cells)16. Also, Li et al. fabricated a 2D SERS substrate consisting of Au NPs and 2D hafnium ditelluride nanosheets for label-free SERS measurements of foodborne pathogenic bacteria17. Recently, carbon dots (CDs), good electron donors, have been used as reductants without other reductants or irradiation to synthesize Au@carbon dot nanoprobes (Au@CDs)18, which have been reported to be efficient materials to enhance SERS activity based on the charge-transfer (CT) effect between Au cores and CD shells19,20. More than that, CDs are recognized as the capping agent and a stabilizer to prevent Au NPs from aggregating21. In addition, it opens up more possibilities for reactions with analytes, as it can provide a large number of binding and active sites20. Taking advantage of the above, Jin et al. developed a fast and controllable method for fabricating Ag@CD NPs with unique SERS properties and excellent catalytic activities for monitoring heterogeneous catalytic reactions in real time18.
Herein, a facile and low-cost method for fabricating core-shell Au@CD SERS substrates to identify cellular components and label-free SERS live cell bioimaging, as well as to detect and differentiate Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was demonstrated, which holds promise for the early diagnosis of disease and a better understanding of cellular processes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x PBS buffer (Cell culture)</td>
			<td>Langeco Technology</td>
			<td>BL316A</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well cell culture plate</td>
			<td>LABSELECT</td>
			<td>11110</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8 (CCK-8)</td>
			<td>GLPBIO</td>
			<td>GK10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Shanghai Aladdin Biochemical Technology</td>
			<td>C108869</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Technologies</td>
			<td>3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant temperature magnetic agitator</td>
			<td>Sartorius Scientific Instruments</td>
			<td>SQP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic high speed centrifuge</td>
			<td>Shanghai Boxun</td>
			<td>SW-CJ-2FD</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose cell culture medium</td>
			<td>Procell</td>
			<td>PM150210</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Sartorius Scientific Instruments</td>
			<td>SQP</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme marker</td>
			<td>Thermo Fisher Technologies</td>
			<td>3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Zhejiang Tianhang Biological Technology</td>
			<td>11011-8611</td>
			<td></td>
		</tr>
		<tr>
			<td>Figure 1</td>
			<td>Figdraw.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fourier infrared spectrometer</td>
			<td>Thermo, America</td>
			<td>Nicolet 380</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>Tecan</td>
			<td>Infinite F50</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>Shanghai Aladdin Biochemical Technology</td>
			<td>G104228</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld Raman spectrometer</td>
			<td>OCEANHOOD, Shanghai, China</td>
			<td>Uspectral-PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>HAuCl4</td>
			<td>Guangzhou Pharmaceutical Company (Guangzhou)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High resolution transmission electron microscope</td>
			<td>Thermo Fisher Technologies</td>
			<td>FEI Tecnai G2 Spirit T12</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature autoclave</td>
			<td>Shanghai Boxun</td>
			<td>YXQ-LS-50S&#160;<img alt="Equation 2" src="/files/ftp_upload/65524/65524eq02.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nanjing Jiangnan Yongxin Optical</td>
			<td>XD-202</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth BR</td>
			<td>Huankai picoorganism</td>
			<td>028320</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical ultra-low temperature refrigerator</td>
			<td>Thermo Fisher Technologies</td>
			<td>ULTS1368</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pancreatin Cell Digestive Solution</td>
			<td>beyotime</td>
			<td>C0207</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin double resistance</td>
			<td>Shanghai Boxun</td>
			<td>YXQ-LS-50S&#160;<img alt="Equation 2" src="/files/ftp_upload/65524/65524eq02.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water meter</td>
			<td>Millipore, USA</td>
			<td>Milli-Q System</td>
			<td></td>
		</tr>
		<tr>
			<td>Raman spectrometer</td>
			<td>Renishaw</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire chip</td>
			<td>beyotime</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic water bath</td>
			<td>Changzhou Noki</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clean table</td>
			<td>Shanghai Boxun</td>
			<td>SW-CJ-2FD</td>
			<td></td>
		</tr>
		<tr>
			<td>Uv-visible light absorption spectrometer</td>
			<td>MADAPA, China</td>
			<td>UV-6100S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire 3.4</td>
			<td>Renishaw</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

label-free surface-enhanced raman scattering bioanalysis based on au@carbon dot nanoprobes

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular fingerprint information of biological samples, as well as its potential in single-cell analysis. This work aims to establish a simple strategy for label-free SERS bioanalysis based on Au@carbon dot nanoprobes (Au@CDs). Here, polyphenol-derived CDs are utilized as the reductant to rapidly synthesize core-shell Au@CD nanostructures, which allows powerful SERS performance even when the concentration of methylene blue (MB) is as low as 10-9 M, due to the cooperative Raman enhancement mechanism. For bioanalysis, Au@CDs can serve as a unique SERS nanosensor to identify the cellular components of biosamples (e.g., cancer cells and bacteria). The molecular fingerprints from different species can be further distinguished after combination with the principal component analysis. In addition, Au@CDs also enable label-free SERS imaging to analyze intracellular composition profiles. This strategy offers a feasible, label-free SERS bioanalysis, opening up a new prospect for nanodiagnosis.

Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes

Fabrication of the Au@CDs is illustrated in Figure 1. The CDs were prepared from CA and GA via a typical hydrothermal process18. Au@CDs were rapidly synthesized by reducing HAuCl4 by CDs in aqueous media at room temperature. The size and morphology of CDs and Au@CDs can be observed by TEM and high-resolution (HR)TEM23. The prepared CDs are monodispersed with small sizes of nearly 2-6 nm (<strong class="xfig"...

Watch this Scientific Journal Video about Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging at JoVE.com

Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging (Video) | JoVE

In this study, we developed a low-cost surface-enhanced Raman scattering (SERS)-based fingerprint nanoprobe with favorable biocompatibility to show label-free live cell bioimaging and detect two bacterial strains, showing in detail how to get SERS spectra of living cells in a non-destructive method.

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular ...