Ultrasensitive fluorescent biosensor for detecting CaMV 35S promoter with proximity extension mediated multiple cascade strand displacement amplification and CRISPR/Cpf 1

Yin Liu; Shiying Zhou; Human Sun; Jiangbo Dong; Liyuan Deng; Na Qi; Yongzhong Wang; Danqun Huo; Changjun Hou

doi:10.1016/j.aca.2022.339973

Ultrasensitive fluorescent biosensor for detecting CaMV 35S promoter with proximity extension mediated multiple cascade strand displacement amplification and CRISPR/Cpf 1

Anal Chim Acta. 2022 Jul 4:1215:339973. doi: 10.1016/j.aca.2022.339973. Epub 2022 May 24.

Authors

Yin Liu¹, Shiying Zhou¹, Human Sun¹, Jiangbo Dong¹, Liyuan Deng¹, Na Qi¹, Yongzhong Wang², Danqun Huo³, Changjun Hou⁴

Affiliations

¹ Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, PR China.
² Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, PR China. Electronic address: wangyzh@cqu.edu.cn.
³ Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, PR China; Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, PR China. Electronic address: huodq@cqu.edu.cn.
⁴ Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, PR China; National Facility for Translational Medicine, Shanghai Jiao Tong University, Shanghai, 200240, PR, China. Electronic address: houcj@cqu.edu.cn.

PMID: 35680336
DOI: 10.1016/j.aca.2022.339973

Abstract

A novel fluorescent biosensor was proposed for detecting the CaMV 35S promoter in genetically modified organisms (GMOs). It was based on a proximity extension mediated multiple cascade strand displacement amplification connected with CRISPR/Cpf 1 (termed PE-MC/SDA-CRISPR/Cpf1). In this protocol, the CaMV 35S was recognized by proximity reaction in the presence of two adjacent primer probes. The proximity extension further triggered the multiple cascade strand displacement amplification (MC/SDA), generating a mass of ssDNA. The products compelled the trans-cleavage activity of CRISPR/Cpf 1, so as to cleave nearby ssDNA-FQ reporters and generate a strong fluorescent signal. The ingenious three-link combination design allowed the CaMV 35S a low background interference. And the MC/SDA combined with CRISPR/Cpf 1 dramatically improved the detection sensitivity. Under optimized conditions, the detection linear range of ultrasensitive fluorescent biosensor for CaMV 35S was from 50 fM to10 pM and 10 pM-500 pM, along with the limit of detection (LOD) down to 14.4 fM. The sensing platform also had excellent performance in the assay of selectivity and real samples. Therefore, the method earned great application potential for transgenic crops.

Keywords: CRISPR/Cpf 1; CaMV 35S; DNA detection; Fluorescence; Strand displacement amplification (SDA).

MeSH terms

Biosensing Techniques* / methods
Clustered Regularly Interspaced Short Palindromic Repeats
Limit of Detection
Nucleic Acid Amplification Techniques* / methods
Promoter Regions, Genetic