Fluolab【JACS】武汉大学黄卫华|微纳传感器首次实现单细胞溶酶体糖苷酶分泌的精准定量监控
文章标题：Nanosensor Quantifying Lysosomal Glycosidase Secretion from Single Living Cells
通讯作者：Wei-Hua Huang (whhuang@whu.edu.cn)
文章概要
引言
免疫系统的稳态平衡对于机体防御病原体入侵和维持内环境稳定至关重要。作为免疫细胞中的核心核心调节效应器，分泌型溶酶体能够在外源刺激下向细胞外释放其内部储存 Bioactive 酶类及蛋白质。当这种分泌过程的酶数量或机制发生异常时，往往会导致细胞废物清除障碍、炎症爆发以及细胞信号通路紊乱，进而诱发自身免疫性疾病、癌症及神经退退行性疾病。因此，实时定量监测分泌型溶酶体酶的动态过程，对于揭示免疫稳态机制和开发新型治疗策略具有深远意义。然而，传统的免疫染色或酶联免疫吸附测定等方法通常需要固定细胞，不仅破坏了细胞活性，且只能提供群体细胞的静态信息。微纳电化学生物传感器虽具备高时空分辨率，但以往的双步酶催化转化方法不仅难以匹配溶酶体分泌的快速动力学，更极易受到细胞内源性活性氧（ROS）的严重干扰。
Figure 1. Schematic representation for the quantitative monitoring of glycosidases. a) Schematic diagram illustrating the hydrolysis of artificial substrates (Glyco-PAPs) by glycosidases leading to the release of the electroactive p-aminophenol (PAP). b) Schematic diagram illustrating the use of a hybrid nanosensor (HNS) to quantitatively monitor glycosidases secreted from lysosomes at a phagocytic cup of a macrophage during the frustrated phagocytosis of a glass nanofiber. The nanopipette of the HNS is utilized to eject Glyco-PAP, while the carbon-coated nanowire electrode (SiC@C NWE) serves to quantitatively monitor the hydrolysis of PAP by glycosidases. c) Scanning electron microscope (SEM) images of a SiC@C NWE; insets: a magnified SEM image of the part marked in the dashed box and transmission electron microscope (TEM) image of a carbon-coated SiC nanowire (SiC@C NW) exhibiting a 200 nm diameter SiC core and a 50 nm thick carbon layer produced by chemical vapor deposition of butane. Scale bars, 2 μm (main image) and 500 nm (insets). d) Repetitive cyclic voltammograms (CVs) recorded at a SiC@C NWE in phosphate-buffered saline (PBS) solution (pH 7.4) either in the absence or presence of β-glucosidase (β-Glu, 10 U), 4-aminophenyl-β-d-glucopyranoside (β-Glu-PAP, 1 mM), PAP (1 mM) and a mixed solution of β-Glu (10 U) and β-Glu-PAP (1 mM) after reacting for 1 min. e) Current statistics of CV signals of the species recorded at +0.6 V in panel d (n = 5 SiC@C NWEs; mean ± s.d.; one-way ANOVA).
主要实验及结论
为了攻克这一瓶颈，研究团队创新性地开发了一种将人工合成底物与混合纳米线-纳米微吸管传感器（HNS）相结合的全新策略。他们精心设计了四种针对不同糖苷酶的人工底物，这些底物通过糖苷键将单糖结构与对氨基苯酚（PAP）相连。当这些非电活性的底物被相应的糖苷酶特异性识别并水解时，能够通过一步催化反应直接释放出具有电活性的外源小分子 PAP，随后在修饰有薄碳层的碳包覆碳化硅纳米线电极（SiC@C NWE）上产生显著的氧化电流信号。实验证实，该传感器在 的最佳电位下具有极佳的定量检测性能和极低的检测限，且由于采用了一步转化产生外源电活性分子的机制，完美避开了细胞高氧化应激环境下内源性 ROS 的干扰。
Figure 2. SiC@C NWEs for quantitative and selective detection of glycosidases with artificial substrates. a) The schematic illustrating 4 glycosidases hydrolytic releasing PAP (indicated by the purple patch) by recognizing the glycosyl portion (shown in green) and hydrolyzing the glycosidic bond (represented by the red bond) within the chemical structure of artificial glycosidase substrates. b, c, d, e) Current calibrations detected by the SiC@C NWEs at +600 mV (vs Ag/AgCl) against a range of Glyco-PAP concentration increments following the reaction with the corresponding α-Glu (b), β-Glu (c), α-Man (d) and β-Gal (e) (1 U), respectively; insets: representative amperometric curves (n = 3 SiC@C NWEs; mean ± s.d.). f, g, h, i) Relative calibration curves showing charge increases of varying activities of α-Glu (f), β-Glu (g), α-Man (h) and β-Gal (i) after reaction with the appropriate Glyco-PAP substrates (10 mM) for 1 min. (n = 3 SiC@C NWEs; mean ± s.d.). j, k, l, m) Normalized plots of currents following the addition of 1 U α-Glu (j), β-Glu (k), α-Man (l) and β-Gal (m) to a given substrate solution (1 mM); currents were normalized to their maximum value after the relevant glycosidase reaction for comparative purposes; insets: representative amperometric curves (n = 3 SiC@C NWEs; mean ± s.d.). All in vitro assays were conducted in phosphate-buffered saline (PBS) at 37 °C and pH 7.4.
Figure 3. Electrochemical detection of simulated glycosidase release using hybrid nanosensors. a) Bright-field micrograph (left) and SEM images (right) of the HNSs; inset: a magnified SEM image of the tip of an HNS marked by the dashed white box. Scale bars, 5 μm (left), 2 μm (right, main image) and 500 nm (right, inset). b) A schematic diagram illustrating the electrochemical detection of glycosidase and the corresponding Glyco-PAP substrates ejected from a micropipette and an HNS, respectively. The micropipette loaded with glycosidases was employed to simulate the secretion of lysosomal glycosidases from a phagocytic cup; solutions α and β representing glycosidases and Glyco-PAP substrates loaded in HNS and micropipette, respectively. c) Amperometric traces for electrochemical detection of the reaction of glycosidases with corresponding Glyco-PAP substrates according to the diagram in panel b. d) Amperometric traces for electrochemical detection of the reaction of solutions α and β loaded in HNS and micropipettes as shown in the schematic diagram in panel b. The green bars in panels b and c indicate duration of each solution α and β ejected from pipettes over 3 s at a pressure of 30 hPa facilitated by a microinjector. e) Corresponding charge statistics for amperometric detection of experiments shown in panels c and d (n = 5 tests; mean ± s.d.; one-way ANOVA).
Figure 4. Characterization of the lysosomes during frustrated phagocytosis of macrophages. a) Distribution of lysosomes in macrophages after staining with CellMask, LysoTracker and Hoechst, either without (M0 type) or with phagocytosis of FITC-labeled glass nanofibers for 12 and 24 h of frustrated phagocytosis. Scale bars, 5 μm (images of M0 and 12 h and main image of 24 h) and 2 μm (enlarged image of the white dashed box of 24 h). b) Expression of LAMP2 in macrophages after staining with Hoechst either without (M0 type) or with phagocytosis of FITC-labeled glass nanofibers for 12 and 24 h of frustrated phagocytosis. Scale bar, 10 μm. c, d) Statistical analysis of the fluorescent intensity for the stained LysoTracker (c) and LAMP2 (d) in panels a and b (n = 30 cells in each 3 groups; data are shown as mean ± s.e.m.; one-way ANOVA). e) Visualization of lysosome migration in macrophages stained with LysoTracker and Hoechst over 2 h period with or without phagocytosis of FITC-labeled glass nanofibers after 12 h of culture. Scale bars, 10 μm.
随后，研究团队引入了巨噬细胞吞噬惰性玻璃纳米纤维的受挫吞噬模型（Frustrated Phagocytosis）。由于长纤维无法被完全包裹和降解，这会诱导细胞形成开放的分泌管腔，促使溶酶体沿轴向轴向迁移并与细胞膜融合，将糖苷酶释放至胞外空间。利用该模型，研究团队首次在活体单细胞水平上成功定量监测了 -葡萄糖苷酶（-Glu）、-葡萄糖苷酶（-Glu）、-甘露糖苷酶（-Man）以及 -半乳糖苷酶（-Gal）四种主要糖苷酶的分泌活性，其中 -Glu 显示出最高的酶活性。更令人兴奋的是，研究团队还通过实时连续监测首次观察到了溶酶体糖苷酶的补充重分泌现象。在首次分泌排空后，巨噬细胞为了消灭难以维持降解的纳米纤维，会启动补偿机制，驱动胞质内的深部溶酶体继续向受挫吞噬口迁移并进行二次释放。统计显示，这种重新分泌的酶活性电荷量约为初始测量值的 。
Figure 5. Hybrid nanosensors for quantitative detection of glycosidases secretion during frustrated phagocytosis. a) Amperometric traces recorded by an HNS located near one cell/nanofiber junction point of a macrophage after Vac-1 incubation without (trace (i) and with ejection of PBS (trace (ii) and β-Glu-PAP (trace (iii) from nanopipette; inset: bright-field micrograph of an HNS positioned near one cell/nanofiber junction point, scale bar, 10 μm. b) Corresponding charge statistic for amperometric detection in panel a (n = 5 macrophages; data are shown as mean ± s.d.; one-way ANOVA). c) Bright-field micrographs of an HNS positioned at a phagocytic cup of a RAW 264.7 macrophage after 12 h of frustrated phagocytosis; scale bar, 20 μm. d) Amperometric traces recorded by HNSs with 10 mM Glyco-PAP ejected from nanopipette while leaving away from (trace (i) and close to (trace (ii) the phagocytic cups of macrophages after 12 h frustrated phagocytosis, respectively. e) Amperometric traces recorded by HNSs with 10 mM Glyco-PAP ejected from nanopipette at the phagocytic cups of macrophages after preincubation of 1-deoxynojirimycin (DNJ, inhibitor of α-Glu), conduritol B epoxide (CBE, inhibitor of β-Glu), swainsonine (Swain, inhibitor of α-Man) or D-galactose (inhibitor of β-Gal), respectively. The green bars in panels a, d and e indicate the duration of each solution ejected from nanopipettes of HNSs within 3 s at 30 hPa by a microinjector. f) Corresponding charge statistics of amperometric detection of glycosidases in panel d and e. The statistical bars for each glycosidase group reflect the charge statistics of HNSs both away from (i) and close to the phagocytic cups of macrophage without (ii) and with (iii) the preincubation of glycosidase inhibitors, respectively (n = 5 macrophages; mean ± s.d.; one-way ANOVA). g) Enzyme activity statistics of the 4 glycosidases in panels f according to the standard curves in Figure 2f–i (n = 5 macrophages; mean ± s.d.; one-way ANOVA).
Figure 6. Supplemental secretion of lysosomal glycosidase during frustrated phagocytosis. a) Amperometric traces recorded by an HNS located away from and close to phagocytic cups of macrophages after 12 h of frustrated phagocytosis with multiple ejections of 10 mM α-Glu-PAP. The cutoffs on the time axis indicate the intervals from the start of the timing of the 2nd ejection of α-Glu-PAP from the nanopipette of the HNS, with the 4th and 5th substrate ejections occurring 20 and 60 min after this timing, respectively. b) Corresponding charge statistics after each α-Glu-PAP ejected from the nanopipette in panel a. c) Localization of lysosomes labeled with LysoTracker and Hoechst for 20 min with and without α-Glu-PAP ejected toward the phagocytic cups after 12 h of frustrated phagocytosis of FITC-labeled glass nanofibers by macrophages. Scale bars, 10 μm (main image) and 1 μm (magnified image). d) A schematic diagram illustrating the electrochemical detection by an HNS of both the original glycosidase and the resecreted glycosidase triggered by the migration of lysosomes from the distal region to the phagocytic cups. e) Amperometric traces of resecretion of β-Glu, α-Man and β-Gal detected by HNSs. The green bars in panels a and e indicate the duration of each 10 mM Glyco-PAPs ejected within 3 s at 30 hPa by a microinjector. f) Corresponding charge statistics for amperometric detection of supplemental glycosidases after multiple ejections of corresponding Glyco-PAPs (n = 5 macrophages; mean ± s.d.; one-way ANOVA). g) Enzyme activity statistics of the resecreted glycosidases in panel f according to the standard curves in Figure 2f–i (n = 5 macrophages; mean ± s.d.; one-way ANOVA).
总结及展望
这项研究成功展示了混合纳米传感器配合特异性人工底物在活细胞单细胞微区进行多靶点、高动态、免受内源物质干扰的定量分析能力。这种创新的检测策略不仅加深了科研人员对巨噬细胞在免疫杀伤过程中精细调节酶活性、维持免疫稳态机制的理解，也具有极强的普适性。未来通过设计更多与目标酶特异性结合并释放电信号的底物，该技术有望拓展到更多种类的溶酶体分泌酶检测中。尽管目前该方法在多目标同时检测时仍依赖于更换不同的传感器，但未来的研究将聚焦于优化界面结构以消除信号交叉干扰，从而推动多通道集成高灵敏度检测系统的进一步发展。