Fig 2: Detection of DCGA response to GzmB.a Schematic illustration of the fluorescence spectral changes of DCGA probe in response to GzmB. b, c The NIR-II fluorescence images (single channel) and corresponding ratio images (b) and fluorescence emission spectra (c) response of DCGA when incubated without or with GzmB (0.2 μM). The “a.u.” means “arbitrary units”. d, e NIR-II fluorescence images (single channel) and corresponding ratio images (d), and ratio values (e) obtained from fluorescence spectra of DCGA incubated with different concentrations of GzmB (0–0.3 μM) (n = 4 independent samples). f NIR-II fluorescence images and corresponding ratio images of DCGA after incubation with different substrates (hydrogen peroxide (H2O2), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), tyrosinase (TYR), cathepsin C (CTSC), neutrophil elastase (NE) and GzmB). g Ratio values were calculated from the images shown in (f) using ImageJ software (n = 4 independent samples). The data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (****p < 0.0001). Source data are provided as a Source Data file.

Fig 3: In vitro detection of DCGA response to GzmB at cellular level.a Schematic representation of the isolation and activation of CD8+ T cells from mouse. b The quantification results of the expression of CD69 (left) and CD25 (right) on CD8+ T cells from flow cytometry (n = 4 independent experiments). c Relative cell viability of BNL CL.2 cells, Hepa 1-6 cells and CD8+ T cells after incubation with DCGA (n = 5 independent experiments). d NIR-II fluorescence images and corresponding ratio images of DCGA after incubation with Hepa1-6 cells, unactivated CD8+ T cells, activated CD8+ T cells, and activated CD8+ T cells pre-treated with the GzmB inhibitor (Ac-IEPD-CHO). e Ratio values calculated from the images shown in (d) using ImageJ software (n = 5 independent experiments). f Schematic representation of DCGA responsive imaging in the co-culture system of activated CD8+ T cells/Hepa1-6 cells. g NIR-II fluorescence images and corresponding ratio images of DCGA after incubation with effector cells (E, activated CD8+ T cells) and target cells (T, Hepa 1-6 cells) at various E:T ratios. h Ratio values calculated from the images shown in (g) using ImageJ software (n = 4 independent experiments). i Secreted GzmB concentrations from cells under different treatments (n = 4 independent experiments). j Correlation between GzmB concentration in cell secretions and the ratio signal (F1060nm/F1525nm) of DCGA in cell pellets under different treatments, analyzed using a linear regression model. k Cell death rate under different treatments was determined by measuring the LDH concentration in cell secretions (n = 4 independent experiments). l The correlation between the cell death rate and the ratio signal of DCGA with different treatments. The treatments included different E:T cell ratios of 0:1, 1:1, 5:1, and 10:1. The data are presented as mean ± SD. Statistical analysis was performed using a two-tailed paired Student’s t-test (for b) and one-way ANOVA followed by Tukey’s multiple comparisons test (for e, h, i, k) (**p < 0.01, ****p < 0.0001). Source data are provided as a Source Data file.

Fig 4: In vivo real-time NIR-II fluorescence imaging of DCGA in Hepa 1-6 tumor model with immunotherapy.a Schematic illustration of timeline for immunotherapy and real-time imaging. b Chemical structures and targets of different immunomodulators for T cell activation and GzmB release. c Representative NIR-II fluorescence images and ratio plots at different time points after i.v. injection of DCGA in tumor-bearing mice treated with different immunomodulators. d, e In vivo NIR-II fluorescence images and ratio plots (d) and the corresponding ratio signal (F1060nm/F1525nm) values (e) of tumor-bearing mice following different treatments at 24 h post-injection of DCGA. f Correlation between the relative tumor volume on day 22 and the ratio signal values of DCGA in tumors on day 10 with different treatments. g Schematic illustration of the correlation between the relative tumor volume and the ratio signal values. The different treatments applied were G1: PBS, G2: NLG919, G3: BMS-1, and G4: BEC, respectively. Source data are provided as a Source Data file.

Fig 5: Synthesis and characterization of DCGA.a Illustration of the preparation of DCGA. b TEM image of DCGA probe (scale bar: 100 nm). Inset: High-magnification TEM image of DCGA (scale bar: 50 nm). The experiments in (b) were repeated independently three times with similar results. c, d DLS results (c) and surface zeta potential (d) results of DCNPs@PEG, DCNPs@GzmB and DCNPs@GzmB-A1094 (termed DCGA) (n = 3 independent samples). e Absorption spectra of GzmB peptide, DCNPs@PEG, A1094 and DCGA. f Overlap of fluorescence emission spectra of DCNPs@GzmB and absorption spectra of A1094. g Fluorescence spectra of DCGA with varying concentrations of A1094 (0–80 μg/mL) under 808 nm laser excitation. h Plot of fluorescence ratio (F1060nm/F1525nm) changes as a function of A1094 concentration (0–80 μg/mL) (n = 3 independent samples). i Average size and PDI changes of DCGA (n = 3 independent samples) probe in medium containing 10% FBS. j Normalized fluorescence changes of DCGA (F1525nm and F1060nm) and small molecule dye IR1061 (F1060nm) under irradiation with 808 nm laser (0.4 W/cm2) for 20 min (n = 3 independent samples). In the figure, the “a.u.” means “arbitrary units”. The data are presented as mean ± SD. Source data are provided as a Source Data file.