An ultrasensitive polydopamine bi-functionalized SERS immunoassay for exosome-based diagnosis and classification of pancreatic cancer

Abstract

Early diagnosis and metastasis monitoring for pancreatic cancer are extremely difficult due to a lack of sensitive liquid biopsy methods and reliable biomarkers. Herein, we developed easy-to-prepare and effective polydopamine-modified immunocapture substrates and an ultrathin polydopamine-encapsulated antibody-reporter-Ag(shell)-Au(core) multilayer (PEARL) Surface-Enhanced Raman Scattering (SERS) nano-tag with a quantitative signal of the Raman reporter at 1072 cm^-1, which achieved ultrasensitive and specific detection of pancreatic cancer-derived exosomes with a detection limit of only one exosome in 2 μL of sample solution (approximately 9 × 10^-19 mol L^-1). Furthermore, by analyzing a 2 μL clinical serum sample, the migration inhibitory factor (MIF) antibody-based SERS immunoassay could not only discriminate pancreatic cancer patients (n = 71) from healthy individuals (n = 32), but also distinguish metastasized tumors from metastasis-free tumors, and Tumor Node Metastasis (TNM) P1-2 stages from the P3 stage (the discriminatory sensitivity was 95.7%). Thus, this novel immunoassay provides a powerful tool for the early diagnosis, classification and metastasis monitoring of pancreatic cancer patients.

Scheme 1. A schematic view of the PDA chip and PEARL SERS tag-based exosome sensors.

Fig. 1. (A) Microscopy images of PDA chips formed with 33 mM dopamine. (B) AFM images of glass slides modified with PDA polymerized with 33 mM dopamine. (C) Raman images of sample points on slides with different modification steps (bare glass, with PDA, with PDA and antibodies) using anti-MIF SERS tags (785 nm excitation, scan area: 5 mm × 5 mm with a 100 μm scan step, and 0.1 s acquisition time for each scan point). (D) High-magnification TEM images of the final SERS tag. (E) SERS spectra of an Au nanoparticle solution (black line) and SERS tag solution (red line). (F) The influence of dopamine concentration on SERS intensity.

Fig. 2. (A) TEM image of HPDE6-C7-derived exosomes. (B) TEM image of PANC-01-derived exosomes. (C) The distribution of the original exosome solution from PANC-01 cells by NTA. (D) Structure and formation of DIO-dyed SM3-P100-antibody-exosome complexes. (E) Relative CD9, CD63, GPC1 and MIF expression from PANC-01- (right side) and HPDE6-C7- (left side) derived exosomes.

Fig. 3. (A) SERS spectra of the anti-CD9, CD63, MIF and GPC1 groups for PANC-01-derived exosomes. (B) Comparison of the intensities at 1072 cm^–1 of the anti-CD9, CD63, MIF and GPC1 groups for PANC-01- and HPDE6-C7-derived exosomes. (C) The Raman spectra (from 1000–1200 cm^–1) for different PANC-01 exosome concentrations (5.44 × 10² to 2.72 × 10¹⁰ particles per mL). (D) Variation of the SERS intensity of MIF at 1072 cm^–1 with PANC-01 exosome concentration. The inset shows the linear relationship between SERS intensity and PANC-01 exosome concentration (5.44 × 10² to 2.72 × 10⁴ particles per mL). For each concentration, the experiments were repeated 3 times. (E) Correlation curve between MIF and PANC-01 exosome concentration using a commercial ELISA kit.

Fig. 4. (A) Shapiro–Wilk analysis plots of the SERS results of serum samples of pancreatic cancer patients (n = 71) and healthy controls (n = 32) using the anti-MIF platform. The ordinate represents log values of Raman intensity; we subtracted the intensity of control samples from experimental samples. (B) Waterfall plots of pancreatic cancer patients (n = 22) and healthy controls (n = 20) using commercial ELISA kit results. The ordinate represents MIF concentrations in pancreatic cancer patients or healthy individuals, and the abscissa represents the number of subjects involved in the study. Shapiro–Wilk analysis plots of the SERS results from 32 healthy individuals and 34 pancreatic cancer patients tested with (C) anti-GPC1 and (D) anti-EGFR platforms.

Fig. 5. (A) Receiver operating characteristic (ROC) curves were calculated for single exosome markers (MIF, GPC-1 and EGFR) (red: pancreatic cancer vs. healthy controls; purple: metastasis vs. non-metastasis; and green: P1–2 vs. P3). AUC stands for the area under the curve. (B) Raman imaging scanning of the 7.2 × 1.8 cm chip containing serum samples from pancreatic cancer patients (P1–8) and healthy individuals (N1–8) tested using the anti-MIF platform.