【JACS】实现79978倍强度增强！新型暴发型近红外化学发光探针助力高灵敏活体成像#

Scheme 1. Schematic Illustration of (a) Previous Design and (b) Our Design; and (c) Synthetic Routes of Chemiluminophores DPD_X_ (X = 2, 3, and 4)#

Figure 1. Photophysical properties of the NIR chemiluminophores. (a) F–-responsive chemiexcitation mechanism of NIR chemiluminophores (DPD_X_ as an example). (b) CL half-life and CL quantum yields of reported NIR chemiluminophores (emission >700 nm) and our chemiluminophores. Details in Table S1. CL spectra (c), intensities (d), and time-courses (e) of DPD1, DPD2, DPD3, and DPD4 (5.0 μM) in DMSO in the presence and absence of TBAF (20 μM). Intensity data are the mean ± SD, n = 3 independent experiments. CL spectra (f) and time-courses (g) of GCL1 and GCL4 (5.0 μM) in DMSO in the presence and absence of TBAF (20 μM). (h) Time-dependent CL visual imaging of GCL1 and GCL4 (10 μM) after addition of TBAF (40 μM) at 37 °C. _t_1/2,CL represents the CL half-life(s) of the corresponding chemiluminophores.#

Figure 2. (a) Chemiexcitation mechanism of DPD_X_ based on Schaap’s adamantyl-phenoxy-1,2-dioxetanes. (b) Summary of the computed activation barrier (Δ_E_a) of the rate-determining step of chemiexcitation for four substituted-adamantyl-phenoxy-1,2-dioxetanes. (c) Relationship between the Δ_E_a and the rate constant of DPD_X_. Detailed DFT calculations are presented in the Supporting Information (Figure S7).#

Figure 3. (a) CL detection of β-gal with DPD4g. (b) CL spectra of DPD4g (10 μM) in the presence and absence of β-gal (1.0 U/mL) in 10 mM PBS (pH = 7.4, 5% DMSO and 10 mM MgCl2). (c) HPLC profile of DPD4g (10 μM) after incubation with β-gal (1.0 U/mL) for 90 min. (d) Time course of the CL intensity of DPD4g (10 μM) incubated with β-gal (1.0 U/mL) in 10 mM PBS (pH = 7.4, 5% DMSO and 10 mM MgCl2). (e) CL changes of DPD4g (10 μM) in the presence of β-gal (1 U/mL), other different enzymes (1.0 U/mL), or β-gal (1.0 U/mL) with an inhibitor (d-galactose, 100 mM) in PBS (10 mM, 5% DMSO and 10 mM MgCl2) at 37 °C. Data are the mean ± SD, n = 3 independent experiments. (f) CL intensities of DPD4g (10 μM) incubated with a function of [β-gal] (0–1.0 U/mL) for 10 min. Data are the mean ± SD, n = 3 independent experiments. (g) CL and FL imaging of HeLa and SKOV3 cells after incubation with DPD4g (10 μM) for 30 min, respectively. SKOV3 + inhibitor group: the SKOV3 cells were treated with an inhibitor (d-galactose, 100 mM) for 1 h before incubation with DPD4g. The blue signal corresponds to the cell nucleus stained with Hoechst 33342, and the red signal corresponds to the cytoplasm treated with DPD4g. (h) Quantification analysis of CL and FL signals in (f). Data are the mean ± SD, n = 3 independent experiments.#

基于性能优异的DPD4母体，研究团队将其进一步构建为特异性响应$\beta$-半乳糖苷酶（$\beta$-gal）的激活型探针DPD4g，用于肿瘤的早期检测。体外实验显示，该探针检测$\beta$-gal的最低检出限低至0.136 mU/mL，且对目标酶具有极高的特异性选择性。在细胞及活体成像实验中，DPD4g展现出极其优异的诊断对比度：在注射入小鼠肿瘤区后，高表达$\beta$-gal的SKOV3肿瘤部位在5分钟内就达到了82.3倍的化学发光信号增强。最终，该探针成功将$\beta$-gal阳性肿瘤与阴性肿瘤清晰地区分开，活体发光强度差异达到15.2倍，完美实现了活体水平的高灵敏度、高对比度肿瘤特异性成像。

Figure 4. Representative CL images (a) and quantification of chemiluminescent signal in the tumor (b) of DPD4g-injected tumor-bearing mice at different time points with or without pretreatment of the inhibitor (d-galactose, 40 μmol/kg). Inhibitor group: SKOV3 tumor-bearing mice were intratumorally injected with d-galactose (40 μmol/kg) 6 h before injecting DPD4g. Two-tailed Student’s t test, SKOV3 vs HeLa, ***p < 0.001 (n = 3, mean ± SD). (c) Histological analysis of tumor sections after different treatments. The blue signal came from the cell nucleus stained with DAPI, and the red signal came from DPD4g.#