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

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

In this research, we developed a low-cost fingerprint nanoprobe, based on surface-enhanced Raman scattering, with favorable biocompatibility. Our probe enables label-free live cell bio-imaging and can detect two bacterial strains by providing molecular fingerprint information. We also conducted a principal component analysis of single living cells.Researchers have recently focused on improving sensitivity, reproducibility, and developing new substrates and technique to provide reliable molecular information. This enables the simultaneous detection of multiple analyses, and it can be combined with machine learning data analyses for improved performance. Obtaining specific rhythmic signals from cells can be challenging, due to the presence of many other chemicals in the sample;which can interfere with the background signal.Compared with the fluorescence detection, the level fluorescence had actual high surface sensitivity. And could realize the directional detection of spacing errors raising cells. More importantly, you can provide the intrinsic chemistry of the at a single cell level in or on the strategy manner under natural conditions.In the future, our work will focus on the complete architecture of carbon-dot based system, including the structure verification and the source We also focus on the biomedical application of this search system such as cell research and the applications.

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 ...

label-free-surface-enhanced-raman-scattering-bioanalysis-based-on

Research

JoVE Journal

Bioengineering

Fabrication of Au@Carbon Dot Nanoprobes

Begin by adding 100 microliters of the prepared carbon dot solution into 200 microliters of chloroauric acid at room temperature for 10 seconds until a purple suspension is formed. Next, centrifuge the purple suspension at 4, 000 G for 10 minutes at room temperature. Gently remove the supernatant using a pipette if removing the gold carbon dot's composite nanoparticles colloids is difficult.Resuspend the gold carbon dot's composite nanoparticles in 200 microliters of deionized water to wash away excess carbon dots. Centrifuge the suspension once again, remove the supernatant, and obtain the core shell nanoparticles by redispersing them in 100 microliters of deionized water. Store the mixture at four degrees Celsius.

Human Epithelial Lung Carcinoma Cells Culture and Cell Surface-Enhanced Raman Scattering (SERS) Experiments

After fabricating the gold carbon dots composite nanoparticles, place a sterile sapphire chip into the 24-well plates and seed cells with one milliliter of the medium into a single well. Incubate the plates in a humid environment to attain 70 to 80%confluence. Add the gold carbon dots composite nanoparticles to the single well and incubate for four to six hours.At the end of the incubation, remove the medium from the sapphire chips. Gently rinse the chips with PBS and dip the sapphire chips in PBS for SERS detection. To perform the SERS experiment, switch on the computer, Raman spectrometer, and 785 nanometer laser.Next, start the accompanying software and perform calibration using silicon wafers. Click new measurement followed by spectral acquisition and adjust the spectrum range. Then go to the acquisition tab, adjust the exposure time and laser power, and click OK to initiate a new spectral acquisition.Use a 20X objective lens for a clear cellular image. Then switch to a 50X lens. After selecting a point on the cells for measurement, click run to initiate the process and then save the spectra.To obtain SERS imaging of A549 cells, click new streamline image acquisition, select the range to be photographed and click OK.Set the edge streamline to 785 nanometers center to 1, 200 centimeter inverse, exposure duration to five seconds, and laser power to 50%The parameter can be modified as required. Click on new of live imaging, choose signal to baseline and set the first limit to 725 and the second limit to 1, 700. Then go to area set up, set the proper steps and click on OK.Finally, click run to start performing SERS imaging.SERS Spectra revealed that even when the concentration of methylene blue is as low as 10 to the minus nine molar, gold carbon dots composite nanoparticles exhibit excellent SERS performance compared to gold nanoparticles. SERS spectra acquired on 20 points exhibited high similarity. One month after the placement of gold carbon dots composite nanoparticles at four degrees Celsius, the average SERS activity at 950, 1, 185 and 1, 620 centimeters inverse showed a degree of decay of about 5.43%11.44%and 13.94%respectively.Gold carbon dots composite nanoparticles barely showed cytotoxicity to A549 cells. The mean spectrum of A549 cells is presented. Gold carbon dots composite nanoparticle aggregates exhibit obvious SERS signals without noise background in the SERS mapping of A549 cells.Spectra from different cell points demonstrated the heterogeneity of components in various cytoplasmic regions.