研究背景与核心痛点#

DNA适配体是一类能够像抗体一样精准识别并结合特定目标分子的单链核酸序列，在生物传感和疾病检测领域被广泛应用。传统的适配体荧光传感器主要依赖于在核酸链上共价标记荧光发光团。然而，这种化学标记方法存在诸多固有缺陷：其合成与纯化成本高昂、工艺复杂，对于超过100个碱基的长链序列几乎难以实现；同时，由于环境干扰或核酸降解，标记传感器极易产生高背景噪音和假阳性信号。

为了克服共价标记的局限，无标记传感策略逐渐成为研究热点。过往的无标记设计多依赖双链DNA或G-四联体（G4）等结构来结合荧光染料。但这些传统DNA结构缺乏特异性的结合口袋，主要依靠非特异性的静电作用和碱基堆积结合染料，容易与样品中的其他非目标核酸发生交叉反应，导致选择性变差、背景信号偏高。

Fig.1 Structure and functional analysis of Lettuce fluorescent light-up DNA aptamer. (a) Modular design strategy and sensing mechanism of Lettuce-based fluorescent sensor. The sensor is composed of three functional modules with specific design requirements (labeled below), including the Lettuce reporter (gray), the transducer (red), and the target-binding aptamer (blue). In the absence of the target (gray hexagon), the sensor remains unfolded. Upon target binding to the aptamer, a change stabilizes the transducer domain, inducing proper folding of Lettuce, which then binds the fluorophore (black) and generates a strong fluorescence signal. (b) Three-dimensional structure model of Lettuce showing stem-loop 1 and fluorophore-binding core. The red box represents the fluorophore-binding core (PDB ID: 8FI2). (c) The secondary structure of Lettuce (left). Stem-loop 1 (boxed in blue) comprises stable fixed sequences from the SELEX library, suggesting it primarily serves as a structural role rather than directly interacting with the fluorophore (yellow). Fluorescence in the presence of three different stem-loop insertions in Lettuce (right). Stem-loop 1 and the replacement stem-loop 1 have nearly identical fluorescence intensities. However, the mismatched stem-loop 1 reduces the fluorescence intensity to baseline even though it retains the original TTC loop. This result indicates that Lettuce is amenable to changes in the sequence of stem-loop 1. However, rigidification at the double-stranded region of stem-loop 1 in the Lettuce structure is necessary to activate the fluorophore.

创新的传感器设计机制#

针对上述难题，本研究利用本团队此前开发的新型荧光发光DNA适配体Lettuce作为核心信号发生模块，构建了一套全新的无标记通用传感平台。与传统的非特异性DNA结构不同，Lettuce自身能够折叠形成精准的三维结构结合口袋，只有当它正确折叠时，才能特异性地结合并“点亮”原本无荧光的游离小分子荧光团。这种基于结构匹配的识别机制极大地降低了背景荧光，提高了传感器的抗干扰能力。

研究团队巧妙设计了一种三模块偶联机制，将Lettuce信号模块、识别目标的特异性适配体模块以及一个起结构调控作用的转换器（Transducer）模块融合在一起。在没有目标分子存在时，整个结构处于不稳定或解折叠状态，Lettuce无法点亮荧光团，系统呈现低背景信号；一旦目标分子出现并与适配体结合，这一结合事件会通过转换器传递，诱导Lettuce正确折叠，从而恢复其结合并点亮荧光团的能力，产生强烈的目标依赖性荧光信号。

Fig.2 Performance of Lettuce-based sensor for small molecule detection. (a) Chemical structure of aflatoxin B1 (AFB1). (b) Optimization of the transducer of the AFB1 sensor. The optimal transducer with the highest signal-to-noise ratio (63-fold) (transducer 1, indicated in the black box) was chosen. (c) Fluorescence response of the AFB1 sensor toward AFB1 with increasing concentration. The linear regression shows excellent correlation (R2 = 0.9979) between fluorescence intensity and AFB1 concentration. (d) Kinetics of fluorescence activation of the Lettuce-based AFB1 sensor. The dashed line indicates 90% of the maximum signal after 10 min. The data are represented as the mean ± SD of three independent repeats. (e) Chemical structure of aflatoxin M1 (AFM1). (f) Optimization of the transducer of the Lettuce-based AFM1 sensor. The optimal transducer (transducer 4, indicated in the black box) with the highest signal-to-noise ratio (5-fold) was chosen. (g) Fluorescence response of the AFM1 sensor toward AFM1 with increasing concentration. The linear regression demonstrates good correlation (R2 = 0.9953) between fluorescence intensity and AFM1 concentration. (h) Kinetics of fluorescence activation of the Lettuce-based AFM1 sensor. The dashed line indicates 90% of the maximum signal after 12 min. The data are represented as the mean ± SD of three independent repeats. (i) Chemical structure of ochratoxin A (OTA). (j) Optimization of the transducer of the Lettuce-based OTA sensor. The optimal transducer domain (transducer 2, indicated in the black box) with the highest signal-to-noise ratio (4.5-fold) was chosen. (k) Fluorescence response of the OTA sensor toward OTA with increasing concentration. The linear regression shows excellent correlation (R2 = 0.9987) between fluorescence intensity and OTA concentration. (l) Kinetics of fluorescence activation of the Lettuce-based OTA sensor. The dashed line indicates 90% of the maximum signal after 30 min. The data are represented as the mean ± SD of three independent repeats.

传感器优化与多目标扩展#

为了实现最优的传感性能，研究团队进行了系统化的三步优化。首先，对转换器的茎环结构、长度及碱基GC含量进行精细调控，确保结构开关在未结合状态下足够稳定，而在结合目标后能高效触发构象转变；其次，通过结构分析和突变筛选，确定了在Lettuce上插入转换器的最佳位置，既保证了荧光核心的高效激活，又避免了天然结构的干扰；最后，优化了目标识别适配体的序列，保留其关键结合口袋。

凭借这一模块化设计，研究团队成功针对小分子、蛋白质及金属离子等多种不同类型的目标物开发了相应的发光传感器。例如，在真菌毒素检测中，针对黄曲霉毒素B1（AFB1）的传感器展示出极高的响应敏感度与特异性；对于蛋白质目标（如凝血酶）以及金属离子（如银离子Ag⁺、锌离子Zn²⁺），传感器均能实现选择性信号激活，显著优于传统G4等无标记检测手段。

Fig.3 High selectivity of Lettuce-based mycotoxin sensors compared with G-quadruplex-based sensors. (a) Selectivity of the Lettuce-based AFB1 sensor. The AFB1 sensor exhibits high specificity toward AFB1 with a 63-fold signal-to-noise ratio. (b) Selectivity of a G-quadruplex-based AFB1 fluorescent sensor. The G4-based sensor employs a “turn-off” mechanism. Target binding to the G-quadruplex disrupts the G-quadruplex: thioflavin T (ThT) complex, resulting in decreased fluorescence intensity. The G4-based AFB1 sensor exhibits low selectivity with only a 3-fold signal-to-noise ratio and similar responses toward AFB1 and AFM1, demonstrating significantly lower selectivity than the Lettuce-based AFB1 sensor (63-fold). (c) Selectivity of the Lettuce-based AFM1 sensor. (d) Selectivity of the OTA sensor. The data are represented as the mean ± SD of three independent repeats.

Fig.4 Lettuce-based paper test strip for AFB1 with high selectivity and rapid response. (a) Schematic illustration of the Lettuce-based paper test strip for AFB1. Upon rehydration with a sample containing AFB1, the sensor binds to AFB1, inducing a conformational change that activates the fluorescence, resulting in a visible color change from dim to bright yellow. The left magnified view shows the sensor in an unfolded state with free fluorophore before target binding. The right magnified view shows the sensor-target complex with activated fluorescence upon AFB1 binding. (b) Dose-response curve of the Lettuce-based test strip toward AFB1 with increasing AFB1 concentration. Test 1 and Test 2 represent independent experimental replicates. The fluorescence images (top) show a concentration-dependent signal increase. The linear regression (bottom) demonstrates good correlation (R2 = 0.9103) between fluorescence intensity and AFB1 concentration. (c) The Lettuce-based AFB1 test strip has good selectivity. Test 1 and Test 2 represent independent experimental replicates. The data indicate that the paper-based sensor exhibits high specificity toward AFB1 with minimal cross-reactivity to other mycotoxins. (d) The stability of the strip after storage for different days. Test 1 and Test 2 represent independent experimental replicates. The sensor maintains good fluorescence response over 21 days, demonstrating good stability over the tested storage period. (e) Fluorescence response of the test strip to AFB1 after storage under different relative humidity conditions (0%, 35%, 55%, and 75% RH). Top: representative fluorescence images of test strips (Test 1 and Test 2); bottom: corresponding normalized fluorescence intensities. (f) Fluorescence response of the test strip to AFB1 after storage at different temperatures (−20, 4, 25, and 37°C). Fluorescence images of test strips represent independent experimental replicates (Test 1 and Test 2, top), corresponding normalized fluorescence intensities at the bottom. Signal intensity remained stable across −20 to 25 °C, while 37 °C resulted in the lowest (84%). Ex = 531 nm, Em = 567 nm.

实际应用与便携式纸基检测#

除了液相检测外，该研究还将此技术成果转化为具有现实应用价值的便携式检测工具。团队开发了一种纸基测试条，用于快速、低成本地检测食品中的小分子真菌毒素。在纸基试纸的制备过程中，通过预先封育与冷冻干燥技术将荧光团与Lettuce传感器固定在滤纸基底上，实现了试剂的长久保存与即开即用。

在针对实际样品（如大米、花生油、牛奶及甘草提取物等复杂基质）的加标回收实验中，该传感系统表现出了优异的抗基质干扰能力与高准确度。实验数据表明，纸纸检测能在短短20分钟内完成显色与成像分析，且检出限完全满足食品安全日常监测的严格标准，展现出在现场快速筛查中的巨大落地潜力。

Fig.5 Lettuce-based sensor for protein and metal ion detection. (a) Optimization of the transducer for the Lettuce-based thrombin sensor. The sensor with the optimal transducer (transducer 4, in the black box) showed a 7-fold increase in fluorescence after thrombin incubation. (b) Dose-response curve of the Lettuce-based thrombin sensor. The linear regression demonstrates good correlation (R2 = 0.9922) between fluorescence intensity and thrombin concentration. (c) Selectivity of the Lettuce-based thrombin sensor. The data indicate a high selectivity of the sensor toward thrombin. (d) Optimization of the Lettuce-based Zn2+ sensor. The Lettuce-based Zn2+ sensor with optimal transducer (transducer 5, in black box) exhibited a 7-fold fluorescence enhancement upon Zn2+ binding. (e) Dose-response curve of the Lettuce-based Zn2+ sensor. The linear relationship (R2 = 0.9723) demonstrates quantitative detection capability. (f) Selectivity of the Lettuce-based Zn2+ sensor. The data show a high selectivity of the sensor toward Zn2+ with minimal cross-reactivity to other metal ions. (g) Optimization of the transducer for the Lettuce-based Ag+ sensor. The Lettuce-based Ag+ sensor with the optimal transducer (transducer 4, in the black box) achieved a 4-fold fluorescence enhancement. (h) Dose-response curve of the Lettuce-based Ag+ sensor. The linear correlation (R2 = 0.9977) demonstrates good quantitative detection. (i) Selectivity of the Lettuce-based Ag+ sensor. The data demonstrate a high selectivity of the sensor toward Ag+. Ex = 531 nm, Em = 567 nm. The data are represented as the mean ± SD of three independent repeats.

研究局限与未来展望#

尽管该Lettuce传感平台展示出了高信噪比、高灵敏度与广谱适用性，但研究中仍存在部分待完善的环节。例如，在面对极度复杂的未处理生物体液或强淬灭性环境时，传感器的荧光响应速率与动力学过程仍有提升空间；此外，纸质试纸在长期保存条件下的热稳定性及大规模生产的一致性控制仍需要进一步测试与优化。

未来的研究方向将聚焦于对Lettuce本身进行结构改造以进一步提升其量子产率与抗环境扰动能力，同时探索将该通用传感平台扩展至更多临床标志物与环境污染物的现场实时检测，推动无标记核酸发光传感技术走向产业化应用。