Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of State Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Life Sciences, Nanjing Forestry University, Nanjing, 210037, China.
Key Lab of Innovative Applications of Bioresources and Functional Molecules of Jiangsu Province, College of Life Science and Chemistry, Jiangsu Second Normal University, Nanjing, 210013, China.
Mikrochim Acta. 2024 Jun 13;191(7):386. doi: 10.1007/s00604-024-06477-z.
The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.
CRISPR/Cas12a 与功能 DNA 的结合为构建用于检测非核酸靶标的生物传感器提供了可能。在本研究中，CRISPR/Cas12a 的激活子中的双链间隔相邻基序 (PAM) 被用作分子开关，并使用在杂交链反应 (HCR) 中与 CRISPR/Cas12a 系统 (APF-CRISPR) 集成的变构探针缀合的 PAM 位点形成构建了灵敏的三磷酸腺苷 (ATP) 检测生物传感器。在没有 ATP 的情况下，含有适体的探针 (AP) 处于茎环结构中，该结构阻止 HCR 的起始。在存在 ATP 的情况下，AP 的结构在 ATP 结合后发生变化，导致 HCR 触发链的释放，并产生具有许多 PAM 位点的长双链 DNA。由于双链 PAM 位点的存在对于触发 CRISPR/Cas12a 的切割活性至关重要，因此 HCR 产物中 PAM 位点的 ATP 依赖性形成可以启动 FQ-报告分子的切割，从而通过测量荧光信号来定量 ATP。通过优化序列元件和检测条件，该适体传感器表现出卓越的检测性能。该测定的检测限 (LOD) 估计为 1.16 nM，其中根据六个重复测量计算空白的标准偏差。检测的动态范围为 25-750 nM，整个测定的工作流程约为 60 分钟。此外，通过将其与用于血清中 ATP 检测的市售化学发光试剂盒进行比较，验证了适体传感器的可靠性和实用性。由于其高灵敏度、特异性和可靠的性能，APF-CRISPR 在用于 ATP 检测的生物分析研究中具有很大的潜力。此外，我们已经提供了一个基于 CRISPR/Cas12a 的适体传感器构建的原理验证，其中 PAM 用于调节 Cas12a 切割活性。
