【JACS】山东师范大学唐波院士团队|“与”逻辑双锁定探针检测血清荧光，预警早期动脉粥样硬化#

Scheme 1. An “AND” Logic Probe PLAQ-SCAN for Simultaneous Sensing of O2•– and Viscosity, and Early Warning of AS#

Figure 1. Mechanistic studies of the “AND” logic Gate-Based probe PLAQ-SCAN. (a) DFT calculations showing the HOMO–LUMO distributions and energy gaps of PLAQ-SCAN and its reaction product PLAQ-OH. (b) Oscillator strengths of PLAQ-OH at planar (0°) and twisted (90°) conformations. (c) Variation of the first excited state energy (S1) of PLAQ-SCAN as a function of the dihedral angle. (d) Oscillator strength of PLAQ-SCAN plotted against the dihedral angle. (e) Variation of the first excited state energy (S1) of PLAQ-OH as a function of the dihedral angle. (f) Oscillator strength of PLAQ-OH plotted against the dihedral angle.#

Figure 2. In vitro detection of O2•–and viscosity by PLAQ-SCAN. (a) UV–vis absorption spectra of probe PLAQ-SCAN in response to O2•– and viscosity. (b) UV–vis absorption spectra of PLAQ-SCAN upon gradual addition of O2•– ranging from 0 to 20 μM. (c) Fluorescence spectra of PLAQ-SCAN in response to O2•– and viscosity. (d) Fluorescence spectra of PLAQ-SCAN (10 μM) with different concentrations of O2•– under high-viscosity conditions. (e) Linear correlation between the fluorescence intensity of PLAQ-SCAN (10 μM) at 675 nm and O2•– concentration under high-viscosity conditions. (f) Fluorescence responses of PLAQ-SCAN (10 μM) to various RNS, ROS, metal ions, and amino acids under high-viscosity conditions. (g) Fluorescence spectra of PLAQ-SCAN (10 μM) with varying viscosities in the presence of O2•– (20 μM). (h) Linear correlation between the fluorescence intensity of PLAQ-SCAN (10 μM) at 675 nm and viscosity in the presence of O2•– (20 μM). (i) Fluorescence responses of PLAQ-SCAN (10 μM) to various RNS, ROS, metal ions, and amino acids in the presence of O2•– (20 μM). Data are presented as mean ± SD (n = 3).#

在细胞实验中，探针因具有较高的亲脂性（测定 $\log P$ 为 3.58），能够特异性靶向定位到巨噬细胞内的脂滴上，与商业化脂滴染料的共定位系数达到 0.96。当用药物单独诱导细胞产生超氧阴离子或单独提升细胞黏度时，荧光均无明显增强。而用脂多糖处理细胞使其同时升高超氧阴离子和黏度后，细胞荧光强度提高了约 2.3 倍。如果在处理过程中加入超氧阴离子清除剂，荧光信号便会大幅下降，这直接证明了荧光激活对双重信号的依赖性。此外，在氧化低密度脂蛋白诱导的泡沫细胞模型中，探针荧光信号提升了 2.45 倍，流式细胞术显示高信号细胞比例从 40.12% 增加到 74.67%。基于探针的特异性，研究团队结合活性蛋白谱技术分析了泡沫细胞内的蛋白质硫醇修饰，发现三羧酸循环和脂质代谢相关通路发生了明显的氧化损伤。

Figure 3. Lipid droplet targeting property of PLAQ-SCAN. (a) Confocal fluorescence imaging of RAW 264.7 cells incubated with different concentrations (5–20 μM) of PLAQ-SCAN, with or without LPS treatment for 24 h. Scale bar: 40 μm. (b) Schematic illustration of the control and experimental groups (with LPS stimulation). (c) Quantitative analysis of relative fluorescence intensity for (a). (d) Co-localization imaging of PLAQ-SCAN with commercial organelle probes in foam cells. The red channel (PLAQ-SCAN) was costained with the green channel (lipid droplet probe BODIPY 493/503, mitochondria probe Mito-Tracker Green, lysosome probe Lyso-Tracker Green) and the nuclear probe Hoechst 33342. The right panels show the corresponding colocalization intensity profiles and Pearson’s correlation coefficients. Scale bar: 20 μm. Data are presented as mean ± SD (n = 3). Significant differences were performed by Student’s t test (***p < 0.001).#

Figure 4. Cellular detection of O2•–and viscosity using PLAQ-SCAN. (a) Fluorescence imaging of RAW 264.7 cells treated with 5 μg/mL PMA (1 h), 10 μM nystatin (1 h), or 20 μg/mL LPS (6 h) and then incubated with PLAQ-SCAN (10 μM). (b) Quantitative analysis of relative fluorescence intensity for (a) (n = 5). (c) Fluorescence imaging of LPS-stimulated cells pretreated with or without Tiron and then incubated with PLAQ-SCAN. (d) Quantitative analysis of relative fluorescence intensity for (c) (n = 5). (e) Schematic illustration of foam cell formation from RAW 264.7 macrophages induced by ox-LDL. (f) Flow cytometry data of PLAQ-SCAN under different treatment conditions. (g) Co-staining imaging of cells with anti-CD36 antibody (red) and Hoechst 33342 (blue). (h) Fluorescence imaging of macrophages and foam cells after incubation with PLAQ-SCAN. (i) Quantitative analysis of relative fluorescence intensity for (g) (n = 3). (j) Quantitative analysis of relative fluorescence intensity for (h) (n = 5). Scale bar: 40 μm. Data are presented as mean ± SD. Significant differences were performed by Student’s t test (ns indicates no significant difference, ***p < 0.001).#

在动物模型实验中，研究团队使用高脂饮食喂养的 $\text{ApoE}^{-/-}$ 小鼠构建动脉粥样硬化模型，并进行了长达 18 周的纵向追踪。关键证据在于，在喂养第 8 周时，提取的离体主动脉组织和外周血清中的荧光信号就已经出现了显著升高，而此时传统的油红 O 染色和 H&E 组织切片均未观察到明显的斑块或脂质沉积。直到第 12 周和第 18 周，组织切片上才逐渐出现肉眼可见的血管壁内膜增厚和大量斑块形成。血清荧光强度的变化趋势与血管病变程度保持了一致性，并且外周血清的检测结果稳定可重复。

Figure 5. Construction of mouse with AS and fluorescence imaging of mouse aortic vessels. C57/Control: C57BL/6J mice with normal diet; ApoE–/–/HFD: ApoE–/– mice with high-fat diet feeding. (a) Schematic illustration of AS progression in ApoE–/–/HFD mice after 18 weeks. (b) Serum lipid levels in C57/Control mice and ApoE–/–/HFD mice for 12 and 18 weeks (TG: triglycerides; T-CHO: total cholesterol; HDL: high-density lipoprotein; LDL: low-density lipoprotein). (c) Nile Red staining of aortic sections from C57/Control mice and 18 week ApoE–/–/HFD mice. Nile Red labels lipid deposition, and DAPI labels nuclei. Scale bar: 100 μm. (d) Quantitative analysis of relative fluorescence intensity for (c). (e) CD36 immunofluorescence staining of aortas from C57/Control and ApoE–/–/HFD mice. Anti-CD36 labels the foam cell marker, and DAPI labels nuclei. Scale bar: 100 μm. (f) Quantitative analysis of relative fluorescence intensity for (e). (g) Fluorescence imaging of aortas from C57/Control mice and ApoE–/–/HFD mice at different time points. (h) Oil Red O staining of aortas from C57/Control mice and ApoE–/–/HFD mice at different time points. Scale bar: 1 cm. (i) Quantitative analysis of fluorescence intensity for (g). Data are presented as mean ± SD (n = 3). Significant differences were performed by Student’s t test (ns indicates no significant difference, *p < 0.05, **p < 0.01).#

Figure 6. Histological Staining and Serum Fluorescence Detection of AS Progression. (a) Schematic workflow for simultaneous detection of superoxide anion and viscosity in the serum of ApoE–/–/HFD mice using PLAQ-SCAN. (b) Representative H & E staining images of the aortic root from C57/Control mice and ApoE–/–/HFD mice at different time points. (c) Representative Oil Red O staining images of the aortic root from C57/Control mice and ApoE–/–/HFD mice at different time points. (d) Fluorescence spectra and quantitative analysis of fluorescence intensity of serum from C57/Control mice and ApoE–/–/HFD mice at different time points after incubation with PLAQ-SCAN. Data are presented as mean ± SD (n = 3). Significant differences were performed by Student’s t test (ns indicates no significant difference, *p < 0.05, **p < 0.01).#