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

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 cell^3,4,5.

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 NPs^4,5. More exhaustive details can be found in these review articles^4,5. 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 imaging^2,11,12,13. 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 shells^19,20. 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.

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.

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 (

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...

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
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
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