Four-stage signal amplification for trace ATP detection using allosteric probe-conjugated strand displacement and CRISPR/Cpf1 trans-cleavage (ASD-Cpf1)

• Published in: Sensors and actuators. B, Chemical Chemical, 2020-11, Vol.323, p.128653, Article 128653
• Main Authors: Wang, Xianfeng, Chen, Xiaolong, Chu, Chengxiang, Deng, Yuanyi, Yang, Mei, Ji, Zhong, Xu, Faliang, Huo, Danqun, Luo, Yang, Hou, Changjun
• Format: Article
• Language: English
• Subjects: Adenosine triphosphate; Allosteric probe; Amplification; ATP; Biosensor; Cleavage; CRISPR; CRISPR/Cpf1; Fluorescence; Interference; Signal processing; Strand displacement amplification
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2020.128653

[Display omitted] •An allosteric probe-conjugated SDA was designed for signal amplification of ATP.•ASD-Cpf1 realizes the detection of non-nucleic acid target.•The programmability makes ASD-Cpf1 become a multipurpose platform for biomolecules measurement.•ASD-Cpf1 provides an ultra-low LOD and a wide liner range for ATP detection. High-performance detection of adenosine triphosphate (ATP) is of great importance due to its irreplaceable roles in various physiological processes. In this work, a four-stage signal amplification was established for ATP detection based on an allosteric probe-conjugated strand displacement amplification (SDA) integrated with CRISPR/Cpf1 system (ASD-Cpf1). An allosteric probe (AH) with a hairpin structure was designed to reduce background interference from self-extension and non-specific amplification during the SDA reaction. ATP bound with the aptamer segment at 5′-terminal of AH probe and subsequently opened the AH probe to activate the SDA reaction. The amplification products were recognized by CRISPR/Cpf1 system and triggered the ssDNase activity of Cpf1, resulting in robust cleavage of surrounding DNA FQ-reporter (fluorophore-quencher labeled short single-stranded DNA sequence). With the four-step amplification, ATP was measured with high sensitivity and low background interference. Under the optimal conditions, a linear relationship was obtained between the fluorescence response and ATP concentrations ranging from 2 pM to 10 μM with a limit of detection (LOD) as low as 1.8 pM. Successful detection of ATP in diluted human serum samples further indicated that ASD-Cpf1 system provided a potential platform for detection of small molecules.