fabrication of au@carbon dot nanoprobes

无标记 SERS 分析：用于分子指纹图谱的 Au@Carbon 点纳米探针

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.

作者聚焦：推进 SERS 技术：用于无标记分析和成像的 Au@Carbon 点纳米探针 

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

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.

Watch this Scientific Journal Video about Fabrication of Au@Carbon Dot Nanoprobes at JoVE.com

Fabrication of Au@Carbon Dot Nanoprobes  

Au@Carbon点纳米探针的制备

首先在室温下将100微升制备的碳点溶液加入200微升氯金酸中10秒，直到形成紫色悬浮液。接下来，在室温下以4， 000G离心紫色悬浮液10分钟。如果难以去除金碳点的复合纳米颗粒胶体，请使用移液管轻轻除去上清液。将金碳点的复合纳米颗粒重悬于200微升去离子水中，以洗去多余的碳点。再次离心悬浮液，除去上清液，将其重新分散在100微升去离子水中得到核壳纳米颗粒。将混合物储存在四摄氏度。

Research

JoVE Journal

Bioengineering

Fabrication of Au@Carbon Dot Nanoprobes

219 次观看.  South China Normal University. 首先在室温下将100微升制备的碳点溶液加入200微升氯金酸中10秒，直到形成紫色悬浮液。接下来，在室温下以4， 000G离心紫色悬浮液10分钟。如果难以去除金碳点的复合纳米颗粒胶体，请使用移液管轻轻除去上清液。将金碳点的复合纳米颗粒重悬于200微升去离子水中，以洗去多余的碳点。再次离心悬浮液，除去上清液，将其重新分散在100微升去离子水中得到核壳纳米颗粒...

视频: Fabrication of Au@Carbon Dot Nanoprobes  

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.

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 ...

科研

生物工程

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.