SERS mapping was performed using a WITec Alpha300 R microspectrometer configured with a 632-nm laser and a highly sensitive electron multiplying charge-coupled device (EMCCD). The laser power of 4 mW and the system frequency were calibrated by the peak of a silicon wafer at ∼520 cm−1. SERS mapping was performed at an area of 60 μm × 60 μm (60 pixels × 60 pixels) with 1-μm spatial resolution using a 20× microscope objective. The SERS spectrum from each pixel was generated with 50-ms integration time. The sample was measured in triplicate; for each replicate, three different positions (left, middle, and right) across the entire channel were scanned. The selected region was scanned to simultaneously detect all four biomarkers, and average SERS spectra were calculated from these spectral images.

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