【JACS】从无色到湛蓝：光化学氧化还原放大技术实现 300 倍高灵敏度小分子免疫检测#

Figure 1. Structures of δ-BLMB and of the BLMB-ZON conjugate. Fluorescence intensity at 695 nm (λexc = 650 nm) after 1 min of 450 nm blue-laser illumination (1.) of 10 μmol L–1 RP3 in the presence of δ-BLMB (50 μmol L–1, ◯) or the BLMB-ZON conjugate (50 μmol L–1, ■), as a function of the additional irradiation time with a 650 nm red diode laser (2.). The initial yellow solutions in aerated ACN (bottom left), due to the RP3 complex, turn blue-green (bottom right) showing the methylene blue formation. Control experiments with RP3 and δ-BLMB after 1 min irradiation with a 450 nm blue laser followed by monitoring in the dark (▲), and with just the 650 nm red laser from t = 0 (◆), are shown as well.#

Figure 2. Proposed redox photocatalytic mechanism for the amplification system. Reactions are grouped in colored boxes according to the conditions under which they occur: blue boxes indicate reactions induced by blue light; reactions into red boxes occur upon red light illumination, and reactions within green boxes correspond to those occurring after the addition of the pro-photocatalyst. Species highlighted in red color appear only under blue light illumination, while the species in brown color are those activated by red light. The blue color highlights methylene blue (MB), the fluorescence of which is monitored as the analytical signal, confirming redox photocatalytic amplification. Added species (photocatalyst and pro-photocatalyst) are shown in black bold typeface.#

Figure 3. Absorption (left) and emission (λexc = 650 nm; right) spectra before (green lines) and after (blue lines) 1 min of 450 nm blue-laser illumination followed by 24 min of 650 nm red-laser irradiation of 50 μmol L–1 δ-BLMB (A) or 50 μmol L–1 BLMB-ZON conjugate (B), both in the presence of 10 μmol L–1 RP3 in ACN. The ZON conjugate exhibited residual MB absorption/emission before illumination, due to some oxidation during its synthesis.#

为了彻底阐明这一高效放大系统背后的根本科学原理，团队展开了深入的电化学动力学与光物理机制研究。光谱与电化学表征结果显示， progressive 添加 $\delta$-BLMB 会显著缩短光催化剂 RP3 的激发态寿命，表现出完美的线性斯特恩-沃尔默（Stern–Volmer）关系，淬灭速率常数高达 $k_q = 2.68 \times 10^9 \text{ M}^{-1}\text{s}^{-1}$，这证实了两者之间存在接近扩散控制极限的高效光诱导电子转移过程。如文章图2和图3的机制模型与吸收/发射光谱所示，整个光催化网络在蓝光诱导下，激发态的 RP3 通过不依赖氧气的路径激活前体生成酰基自由基和 $\text{MB}^{-}$ 中间体。在随后的红光激发下，原位新生成的痕量 $\text{MB}^{+}$ 进一步作为次级催化剂参与自发性氧化还原循环。有趣的是，脱氧环境下的系统放大效率甚至略高于有氧环境，这进一步印证了该机制是基于光诱导电子转移而非活性氧（ROS）介导的通路。此外，尽管 ZON 缀合物中的酚羟基具有一定的自由基清除效应，使得其显色和荧光增强速度略慢于纯 $\delta$-BLMB，但两者在红光循环下均展现出了极其显著的特征吸收带与荧光跃迁。

Figure 4. (A) Scheme of the photoamplified assay in heterogeneous medium (magnetic beads/microplate). (B) Fluorescence of MB at 695 nm using 1 μmol L–1 BLMB-ZON conjugate, in the presence (+ZON) and in the absence (0) of 10 μmol L–1 ZON, according to the assay described in panel (A) (n = 3); (i) immediately after 5 min of blue-LED irradiation and addition of 153 μmol L–1 δ-BLMB (pro-photocatalyst); (ii) after subsequent red-diode laser illumination for 7 min.#

最终，研究团队成功将这一先进的光化学放大协议集成到了针对霉菌毒素 ZON 的异质免疫分析应用中。如文章图4所示，该方法完美耦合了生物分子识别与光驱动氧化还原循环，在磁珠和微孔板平台中构建了竞争性抑制免疫测定模式。游离的 ZON 毒素与 BLMB-ZON 缀合物共同竞争结合特异性单克隆抗体（Ab#4），在洗涤并进行溶剂交换后加入光催化剂 RP3。在自主研发的 96 孔板蓝/红双色 LED 照明装置下，系统首先通过蓝光照射启动初始催化并产生极微量的亚甲基蓝前体，随后加入作为“前催化剂”的 $\delta$-BLMB 并投射红光进行级联放大。实验数据令人振奋地表明，通过这种巧妙的级联接力，系统的光化学放大倍数达到了惊人的 300 倍。在理想的优化条件下，该系统甚至有望实现高达 3 个数量级（1000倍）的灵敏度跃升，成功实现了对微量毒素的高特异性、超灵敏荧光定量检测。