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JoVE Journal Bioengineering

Bioengineering

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

Published: June 9, 2023 doi: 10.3791/65524
Automatic Translation
1, 1, 1, 1, 1, 1, 1, 1
1MOE Key Laboratory of Laser Life Science & Guangdong Provincial Key Laboratory of Life Science, College of Biophotonics, South China Normal University

Summary

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.

Abstract

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.

Introduction

Single-cell analysis is essential for the study of revealing cellular heterogeneity and assessing the comprehensive state of the cell. The cell'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'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. 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 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.

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Protocol

1. Fabrication of Au@CDs

NOTE: Figure 1 illustrates a fabrication procedure for Au@CDs.

  1. Prepare CD solution using citric acid (CA) and gallic acid (GA) via a typical hydrothermal treatment procedure18. Add 100 µL of 3.0 mg mL-1 of the prepared CD solution into 200 µL of 10 mM chloroauric acid (HAuCl4) (see Table of Materials) at room temperature for 10 s until a purple suspension is produced.
  2. Centrifuge the purple suspension at 4,000 × g for 10 min at room temperature and gently remove the supernatant using a pipette if removing the Au@CD colloids is difficult.
  3. Resuspend the Au@CDs with 200 µL of deionized water (resistivity of 18.2 MΩcm) to wash the excess CDs.
  4. Repeat step 1.2 and obtain the core-shell NPs, redispersed in 100 µL of deionized water, and store at 4 °C. The NPs can be stored for 1 month.

2. Characterization of Au@CDs

  1. Transmission electron microscopy (TEM)
    1. Leave the CD and Au@CD samples overnight at -80 °C to lyophilize from a frozen solution and crush after lyophilization into powder.
    2. Disperse the obtained powder samples into the deionized water adequately by sonication, at 100% power, for 5 min.
    3. 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).
  2. Fourier transform infrared spectroscopy (FT-IR)
    1. 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.
  3. Ultraviolet-visible-near infrared (UV-vis-NIR) absorbance spectroscopy
    1. 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.
  4. Raman spectra
    1. 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.
    2. Add an equal volume of prepared Au@CDs samples into 5 µL of methylene blue (MB) solution and thoroughly mix.
    3. Place a drop of suspension onto the brass substrate to collect the SERS spectra.

3. Cell culture

  1. Culture human epithelial lung carcinoma cells (A549 cells, passaged in the laboratory) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and incubate in a humidified incubator at 37 °C with 5% CO2.
  2. Seed the cells in 96-well plates (1 × 105 cells/well) and treat with Au@CDs in different concentrations in the range of 20-100 µM. Use a CCK-8 assay kit to measure the cell viability (see Table of Materials). Three independent duplicates are used in each treatment.
  3. For performing the SERS experiments, proceed following the steps below:
    1. 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 °C with 5% CO2overnight to reach 70%-80% confluence.
    2. 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.
    3. 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.
    4. Remove the medium, gently rinse the sapphire chips with phosphate-buffered saline (PBS), and dip them in PBS for SERS detection16.

4. Cell SERS experiments

  1. Open the computer, switch on the Raman spectrometer, start the software and then switch on the 785 nm laser (see Table of Materials).
  2. Click on New Measurement and start a new spectral acquisition. Calibrate17 with silicon wafers before sample measurement.
  3. 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.
  4. 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.
  5. Save the spectra.
  6. 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%.
  7. 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.

5. Bacterial culture and SERS measurements

  1. 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 °C to A600 0.4 to 0.6, centrifuge the culture at 5,000 × g for 5 min to pellet the cells.
  2. Resuspend the pellets in 1 mL of PBS followed by a 5 min 5,000 × g centrifugation (at room temperature) twice.
  3. Mix the bacterial culture with the Au@CDs and observe the mixture directly under the SERS platform.

6. Data analysis

  1. Smooth the spectra and correct the baseline.
  2. Perform principal component analysis (PCA)16 with the processed data.

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Representative Results

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 (Figure 2A). The spherical Au nucleus is coated with a layer of CD shell of about 2.1 nm (Figure 2B,C and Supplementary Figure 1).

To confirm the structures of CDs and Au@CDs, FT-IR spectra were recorded to analyze the organic functional groups (Figure 2D). The band at 3,000-3,600/2,500-2,800 cm-1, 1,251/1,629 cm-1, and 1,471 cm-1 can be assigned to O-H stretching vibration, C=O stretching vibration, and C-O stretching vibration, respectively24,25. Also, the stretching vibrational band at 1,741 cm-1 is related to C-C24. A few of these functional groups from GA and CA are also present in CDs and Au@CDs, suggesting the successful synthesis of NPs. UV-vis absorption spectra of CDs have a characteristic peak at 265 nm, indicating an isolated aromatic structure in the carbon cores26, which disappears upon reduction. The spectrum of Au@CDs exhibits a characteristic peak at 545 nm, which is associated with the SPR absorption band of the Au nucleus, indicating fabrication of the composite nanostructure (Figure 2E).

As shown in Figure 3A,B, even when the concentration of MB is as low as 10-9 M, Au@CDs exhibit excellent SERS performance compared to Au NPs. The enhancement factor (EF) can be calculated by the following equation16, where ISERS and I0 refer to the Raman intensities of the SERS spectrum and normal Raman, respectively, and CSERS and C0 refer to the concentrations of the substrate molecules used for SERS and Raman measurements, respectively. Taking 1,620 cm-1 of MB to calculate, the EF of Au@CDs is nearly 2.1 × 105, which is about three times stronger than that of the Au NPs (6.8 × 104).

Equation 1

A few characteristic peaks can be detected, such as 770 cm-1 (in-plane bending of C-H), 1,398 cm-1 (symmetric stretching of C-N), and 1,625 cm-1 (C-C ring stretching), which is consistent with reported literature27. At the same time, 10-5 M to 10-9 M of MB are detected, taking the SERS band at 1,620 cm-1 for quantification; the linear relationship is defined as y = 0.2437x + 2.1756 (R2 = 0.9922) (Figure 3B). In addition to sensitivity, reproducibility and long-term stability are both significant indicators for SERS substrates. Therefore, SERS spectra were acquired on 20 points randomly, exhibiting high similarity among them (Figure 3C,D); four spectra were collected during 1 month, the characteristic peaks of MB were still detected 1 month after placement at 4 °C, and the average SERS activity at 950 cm-1, 1,185 cm-1, and 1,620 cm-1 only showed a degree of decay of about 5.43%, 11.44%, and 13.94%, respectively (Figure 3E), indicating the good reproducibility and long-term stability of Au@CD substrates.

In the present cell SERS measurements, firstly, the cytotoxicity of the NPs was tested. Au@CDs (20-100 µM) barely showed cytotoxicity to A549 cells (Figure 3F). The above results suggest the high potential of Au@CDs to be applied to label-free SERS measurements in living cells. As shown in Figure 4, the Au@CD-assembled SERS substrate provides integrated SERS mapping of the cellular components from 800 to 1,700 cm-1. It has been observed that Au@CD NP aggregates exhibit obvious SERS signals without noise background in the SERS mapping of A549 cells (Figure 4B). Three spectra from different cell points are shown in Figure 4C, demonstrating the heterogeneity of components in various cytoplasmic regions and the superiority of the Au@CDs as SERS probes for single-cell analysis. The mean spectrum of A549 cells is shown in Figure 4A, and abundant cellular information can be observed. Detailed characteristic peak assignments are provided in Table 1.

In addition, Au@CDs also demonstrate a good ability to detect and differentiate two bacterial strains. The spectra obtained are quite similar to the published literature for E. coli and S. aureus, such as phenylalanine at 1,030 cm-1 and a ring breathing of nucleic acid at 741 cm-1 (Figure 5A, B), which verifiesthe reliability of SERS measurements28. Detailed characteristic peak assignments28,29,30,31 are provided in Table 2. The PCA model also discriminates well (Figure 5C,D).

Figure 1
Figure 1: Schematic illustration of the synthesis pathway of the Au@CDs. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Representative characterization of CDs and Au@CDs. (A) HRTEM image of CDs. Scale bar: 10 nm. (B) TEM image of Au@CDs. Scale bar: 100 nm. (C) HRTEM image of the interface region of Au@CDs. Scale bar: 10 nm; inset: 2 nm. (D) FT-IR spectra of raw GA, CA, and as-prepared CDs and Au@CDs. (E) UV-vis absorption spectra of CDs, Au NPs, and Au@CDs. Please click here to view a larger version of this figure.

Figure 3
Figure 3: SERS activities of Au@CDs. (A) SERS spectra of bulk MB (10-5 M, black line) and the 10-9 M MB (blue line and green line) solution. (B) SERS spectra of MB at different concentrations (10-9-10-5 M). Insert: The linear relationship of the SERS band at 1,620 cm-1 of MB, y = 0.2437x + 2.1756 (R2 = 0.9922). Results of SERS substrate reproducibility (C), relative standard deviation (RSD) histogram (1,620 cm-1 peak of MB at 10-7 M) (D), and long-term stability (E) experiments. (F) Cell viabilities of A549 cells after 24 h of incubation with the Au@CD concentration gradient. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Fingerprint analysis and label-free SERS imaging of A549 cells. (A) Mean SERS spectrum of A549 cells. (B) The bright field, SERS mapping, and merged images of the A549 cells. Scale bars: 20 µm. (C) SERS spectra of different cell points marked in the merged image of 1, 2, and 3. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Fingerprint analysis of two bacterial strains. The mean SERS spectra of E. coli (A)and S. aureus (B). The 2D PCA (C) and 3D PCA (D) on the differential analysis of two bacterial strains. Please click here to view a larger version of this figure.

Table 1: Assignments for the Raman peaks of A549 cells. Abbreviations: Pro = proline; HYP = hydroxyproline; Tyr = tyrosine; Trp = trypotophan; Phe = phenylalanine; A = adenine; T = thymine; C = cytosine; G = guanine; bend = bending; str = stretching; def = deformation; twist = twisting; breath = breathing; wag = wagging; sym = symmetrical; asym = asymmetrical; bk = backbone. Please click here to download this Table.

Table 2: Assignments for the Raman peaks of E. coli and S. aureus. Abbreviations: Tyr = tyrosine; Phe = phenylalanine; A = adenine; G = guanine; str = stretching; def = deformation; breath = breathing; sym = symmetrical. Please click here to download this Table.

Supplementary Figure 1: Dynamic light scattering study of Au@CDs. Please click here to download this File.

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Discussion

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 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 chemical enhancement between Au NPs and CDs.

There are a few key points during SERS measurements in the previous protocol. Firstly, before Au@CDs are applied in cells and bacterial samples, it's necessary to ensure a superior SERS substrate formula as well as the right concentration, which can be verified by dye molecules. Subsequently, the SERS spectrum is inconsistent at every cellular point because the biological samples are complex and dynamic.

If taking a bigger laser spot, the SERS spectrum from different points will be more similar. Of note, the SERS spectrum of the same cellular point may not be in accordance with different laser power. It's well accepted that the average SERS spectrum of biological samples is consistent; therefore, it's critical to obtain the SERS spectrum in the same cellular regions as much as possible and then to average several different cellular regions. In addition, intracellular SERS detection sensitivity is easily affected by background signals. The Raman background signals mainly come from three aspects, including (1) a substrate, such as sapphire, silicon wafer, and so on, (2) a SERS probe, and (3) biological samples. Consequently, it is recommended to choose substrates without background noise whenever possible, although this is difficult.

Compared to conventional Raman spectroscopy, SERS can substantially improve the signal intensity by several orders of magnitude33. In this work, gold was chosen for the fabrication of composite NPs because it possessed a better ability to optimize the size and shape, being less cytotoxic as well as more chemically inert and robust than silver34. Meanwhile, CD shells attached to Au surfaces can reduce the interactions between cells and metal NPs33. In conclusion, the above results demonstrated label-free SERS as a noninvasive and non-destructive technique, which can provide an intrinsic chemical composition of analytes on a single-cell level35.

Previous studies have shown that it is essential to control the appropriate detection environment and time in case of distortion of native fingerprint information5. Therefore, it's essential to make sure there is no damage on the cells, with the aim of obtaining reliable spectra. Other challenges include: (1) for intracellular SERS measurements, SERS-active Au NPs may interact with intracellular components such as proteins, lipids, or nucleic acids4. Therefore, biocompatibility and SERS signal assignments need to be considered; (2) constant laser exposure during SERS detection may damage the cell cytotoxicity or biosamples; and (3) as shown in Figure 5A,B, the portable SERS platform still has some limitations-either Raman intensity or signals that can be detected is not good as what is shown in Figure 4.

In addition, when combined with other technologies such as droplet microfluidics36, deep learning37, machine learning38, catalytic hairpin assembly amplification technology39, and fluorescence technology40, these integrated strategies can develop the superiority of SERS.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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).

Materials

Name Company Catalog Number Comments
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 Equation 2
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 Equation 2
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

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References

  1. Zenobi, R. Single-cell metabolomics: analytical and biological perspectives. Science. 342 (6163), 1243259 (2013).
  2. 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).
  3. 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).
  4. Langer, J., et al. Present and future of surface-enhanced Raman scattering. ACS Nano. 14 (1), 28-117 (2020).
  5. Zong, C., et al. Surface-enhanced Raman spectroscopy for bioanalysis: reliability and challenges. Chemical Reviews. 118 (10), 4946-4980 (2018).
  6. 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).
  7. 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).
  8. 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).
  9. 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).
  10. 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).
  11. 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).
  12. 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).
  13. 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).
  14. 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).
  15. 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).
  16. 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).
  17. 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).
  18. 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).
  19. 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).
  20. 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).
  21. Wang, X., et al. Reduced state carbon dots as both reductant and stabilizer for the synthesis of gold nanoparticles. Carbon. 64, 499-506 (2013).
  22. Zhu, M., et al. Physicochemical properties determine nanomaterial cellular uptake, transport, and fate. Accounts of Chemical Research. 46 (3), 622-631 (2013).
  23. 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).
  24. Fiori, F., et al. Highly photostable carbon dots from citric acid for bioimaging. Materials. 15 (7), 2395 (2022).
  25. 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).
  26. 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).
  27. 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).
  28. Efrima, S., et al. Understanding SERS of bacteria. Journal of Raman Spectroscopy. 40 (3), 277-288 (2009).
  29. Movasaghi, Z., Rehman, S., Rehman, I. U. Raman spectroscopy of biological tissues. Applied Spectroscopy Reviews. 42 (5), 493-541 (2007).
  30. 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).
  31. 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).
  32. 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).
  33. 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).
  34. 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).
  35. Weiss, R., et al. Surface-enhanced Raman spectroscopy of microorganisms: limitations and applicability on the single-cell level. Analyst. 144 (3), 943-953 (2019).
  36. Oliveira, K., et al. Multiplex SERS phenotyping of single cancer cells in microdroplets. Advanced Optical Materials. 11 (1), 2201500 (2023).
  37. Ho, C. S., et al. Rapid identification of pathogenic bacteria using Raman spectroscopy and deep learning. Nature Communications. 10 (1), 4927 (2019).
  38. Spedalieri, C., Kneipp, J. Surface enhanced Raman scattering for probing cellular biochemistry. Nanoscale. 14 (14), 5314-5328 (2022).
  39. 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).
  40. 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).

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Nanoprobe Biocompatibility Live Cell Bio-imaging Bacterial Detection Molecular Fingerprint Information Principal Component Analysis Sensitivity Improvement Reproducibility Substrates Technique Development Machine Learning Data Analysis Rhythmic Signals Fluorescence Detection Surface Sensitivity Directional Detection Single Cell Level Analysis Carbon-dot Based System Structure Verification Biomedical Applications
Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes
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Zheng, Y., Xiao, X., Li, Z., Shao,… More

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).

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