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 until a purple suspension is produced.</li>
	<li>Centrifuge the purple suspension at 4,000 &#215; g for 10 min at room temperature and gently remove the supernatant using a pipette if removing the Au@CD colloids is difficult.</li>
	<li>Resuspend the Au@CDs with 200 &#181;L of deionized water (resistivity of 18.2 M&#8486;cm) to wash the excess CDs.</li>
	<li>Repeat step 1.2 and obtain the core-shell NPs, redispersed in 100 &#181;L of deionized water, and store at 4 &#176;C. The NPs can be stored for 1 month.</li>
</ol>
2. Characterization of Au@CDs
<ol>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Leave the CD and Au@CD samples overnight at -80 &#176;C to lyophilize from a frozen solution and crush after lyophilization into powder.</li>
		<li>Disperse the obtained powder samples into the deionized water adequately by sonication, at 100% power, for 5 min.</li>
		<li>Drop a few drops of the sample suspension onto a Cu-coated TEM grid covered with a lacey carbon film, and capture images after drying using a transmission electron microscope at a 200 kV acceleration voltage (see Table of Materials).</li>
	</ol>
	</li>
	<li>Fourier transform infrared spectroscopy (FT-IR)
	<ol>
		<li>Repeat step 2.1.1 to obtain power samples, grind a few pre-dried KBr particles into powders in a mortar, add a pinch of the sample, and mix with the KBr powders simultaneously for IR characterization16.</li>
	</ol>
	</li>
	<li>Ultraviolet-visible-near infrared (UV-vis-NIR) absorbance spectroscopy
	<ol>
		<li>Prepare the suspension of CDs and Au@CDs with proper concentration to ensure the absorbance is less than one and perform UV-vis-NIR characterization16.</li>
	</ol>
	</li>
	<li>Raman spectra
	<ol>
		<li>Switch on the 785 nm semiconductor laser, with a laser power of 10 mW and objection magnification of 20x. Set the exposure duration to 5 s and accumulations number to three.</li>
		<li>Add an equal volume of prepared Au@CDs samples into 5 &#181;L of methylene blue (MB) solution and thoroughly mix.</li>
		<li>Place a drop of suspension onto the brass substrate to collect the SERS spectra.</li>
	</ol>
	</li>
</ol>
3. Cell culture
<ol>
	<li>Culture human epithelial lung carcinoma cells (A549 cells, passaged in the laboratory) in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and incubate in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Seed the cells in 96-well plates (1 &#215; 105 cells/well) and treat with Au@CDs in different concentrations in the range of 20-100 &#181;M. Use a CCK-8 assay kit to measure the cell viability (see Table of Materials). Three independent duplicates are used in each treatment.</li>
	<li>For performing the SERS experiments, proceed following the steps below:
	<ol>
		<li>Put a sterile sapphire chip (see Table of Materials) into the 12-well plates and then seed the cells onto a single well with 1 mL of medium. Incubate the plates in a humidified incubator at 37 &#176;C with 5% CO2overnight to reach 70%-80% confluence.</li>
		<li>Add the Au@CDs into the single well and incubate for 4-6 h. 
		NOTE: Before incubation, test the stability of substrates, especially solution phase NPs. These materials are susceptible to degradation through dissolution, aggregation, and sedimentation processes during storage and use, which may decrease EM losses and decrease SERS activity. More significantly, for cellular uptake, it could be largely affected by the size of the NPs22.</li>
		<li>During the incubation, substrates will enter the cells through endocytic uptake. After incubation, observe the cells under an optical microscope until a few black particles are seen inside the cells. 
		NOTE: If the SERS intensity is weak, try to enhance the Au@CD concentration or prolong the incubation time.</li>
		<li>Remove the medium, gently rinse the sapphire chips with phosphate-buffered saline (PBS), and dip them in PBS for SERS detection16.</li>
	</ol>
	</li>
</ol>
4. Cell SERS experiments
<ol>
	<li>Open the computer, switch on the Raman spectrometer, start the software and then switch on the 785 nm laser&#160;(see Table of Materials).</li>
	<li>Click on New Measurement and start a new spectral acquisition. Calibrate17&#160;with silicon wafers before sample measurement.</li>
	<li>Take a 20x objective lens to ensure a clear cellular image is observed, then change the objective lens to 50x. Set the laser power from low to high and the appropriate exposure time and accumulations. 
	NOTE: Before SERS measurement, check the appropriate laser power to prevent irreversible cell damage and ensure that the acquisition parameters are consistent. SERS intensity is related to laser power, spot size, accumulation, and exposure time.</li>
	<li>Choose a point of cells to measure and click on Run. Each cell is measured by 20 spots, and 10 cells are measured to take the average. 
	NOTE: Intracellular components are complicated, leading to wide spectra disparity. Therefore, take spectra in similar cellular regions and take as many spectra as possible.</li>
	<li>Save the spectra.</li>
	<li>Click on New Streamline image acquisition, then click on Video Review, select the range to be photographed, and click OK. Set the parameters: 785 nm edge streamline, centre of 1,200 cm-1, exposure duration of 5 s, accumulations number of three, and laser power to 50%.</li>
	<li>Click on New of Living imaging, choose Signal to Baseline, set the range from first limit 625 to second limit 1,700, and then click on Area setup. Then set the proper steps, and click on Apply and OK. Finally, click Run to start performing SERS imaging.</li>
</ol>
5. Bacterial culture and SERS measurements
<ol>
	<li>Dilute overnight cultures of E. coli and S. aureus (1:100) in 5 mL of the new Luria-Bertani (LB) medium (see Table of Materials). After growth at 37 &#176;C to A600 0.4 to 0.6,&#160;centrifuge the culture at 5,000 &#215; g for 5 min to pellet the cells.</li>
	<li>Resuspend the pellets in 1 mL of PBS followed by a 5 min 5,000 &#215; g centrifugation (at room temperature) twice.</li>
	<li>Mix the bacterial culture with the Au@CDs and observe the mixture directly under the SERS platform.</li>
</ol>
6. Data analysis
<ol>
	<li>Smooth the spectra and correct the baseline.</li>
	<li>Perform principal component analysis (PCA)16&#160;with the processed data.</li>
</ol>

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

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The authors have nothing to disclose.

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.

Author Spotlight: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging 

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

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 ...

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JoVE Journal

Bioengineering

1.1K Views.  South China Normal University. 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.