## 2 Materials and Methods
### SERS Measurement and Analysis  
The SERS substrate used in this study was silver colloid, which was prepared based on a classical sodium citrate reduction method [41]. First, 100 mL of \(10^{-3}\) mol/L silver nitrate solution (Carbon Twelve Regent, Shenzhen, China) and 2.5 mL sodium citrate solution (Macklin Regent, Shanghai, China) with a mass fraction of 1% were prepared. Then, the silver nitrate solution was heated and stirred continuously until boiling. The sodium citrate solution was added, and the solution was boiled for 1 h. The colloidal solution was kept away from light at room temperature. The UV-vis spectrum and a transmission electron microscope (TEM, FEI, Hillsboro, OR, U.S.) image of the silver colloid is displayed in Figure 1. More TEM images and the size distribution measurement of the silver colloid are reported in the Supplementary Materials (Figures S1 and S2). The averaged silver nanoparticle size was measured to be 64 nm.  
Throughout the study, SERS spectra of individual cells were collected with a confocal Raman spectrometer (LabRAM Xplora Plus, HORIBA, France) equipped with a 50\(\times\) objective, a charge-coupled device (CCD) camera, and a 638 nm laser with around 10 mW power at sam  
Figure 1: (**a**) The UV–vis absorption spectrum of the surface-enhanced Raman spectroscopy (SERS) substrate, (**b**) the TEM image of silver nanoparticles.  
of the spectrometer was 400-1700 cm\({}^{-1}\) with a spectral resolution of 1 cm\({}^{-1}\). The integration time for acquiring each SERS spectrum was 5 s. Before collecting the SERS spectra of cells, 2 \(\upmu\)L silver colloid was dropped into the culture dishes and the cells were re-incubated for 3 h in culture medium containing the silver colloid. Then, the cells were washed in PBS three times and 20 independent cells from each of the groups (control group, cordycepin-12 h group and cordycepin-48 h group) were measured with the SERS respectively. Therefore, a total of 60 spectra were obtained.  
Necessary postprocessing steps were applied to optimize and analyze the acquired SERS spectra. First, the narrow cosmic rays were removed, and the baseline of the individual spectrum was corrected with a fifth-order polynomial fitting program to correct the fluorescence background. Both operations were performed in LabSpec 6 software (HORIBA Scientific, Paris, France, 2017). Next, all SERS spectra were smoothed by the nine-point Savitzky-Golay method and normalized based on the highest peaks. The 20 spectra from each of the experimental groups (cordycepin-12 h group and cordycepin-48 h group) were averaged to acquire a single averaged spectrum, which was then used to perform subtractions with the averaged spectrum of the control group. To further elucidate apoptosis-induced changes over time, intensity ratios between the apoptotic groups (cordycepin-12 h group, cordycepin-48 h group) and the control group were calculated at several important characteristic peaks. The results were displayed in a bar chart. Furthermore, principal component analysis (PCA) was performed on all 60 acquired spectra. The spectra were normalized. No smoothing was applied. PCA is a multivariate statistical analysis method that converts multidimensional variables in high-dimensional space into a small number of comprehensive new variables, so as to realize signal feature extraction and analysis [42]. In this study, PCA was used to differentiate the cordycepin-12 h group, the cordycepin-48 h group, and the control group.  
In addition, the spectrum of cordycepin was measured using SERS (Supplementary Figure S3). It served as the proof that signal changes in different cell groups were not from the enhanced signals of cordycepin. Experiments were conducted to verify that the SERS substrate, i.e., silver nanoparticles, did not induce HeLa cell apoptosis. The methods, results, and figures (Supplementary Figures S4 and S5) of this experiment are presented in supplementary materials.