Target-triggered triple isothermal cascade amplification strategy for ultrasensitive microRNA-21 detection at sub-attomole level.
MicroRNA-21 (miR-21) is a promising diagnostic biomarker for breast cancer screening and disease progression, thus the method for the sensitive and selective detection of miR-21 is vital to its clinical diagnosis. Herein, we develop a novel method to quantify miR-21 levels as low as attomolar sensitivity by a target-triggered triple isothermal cascade amplification (3TICA) strategy. An ingenious unimolecular DNA template with three functional parts has been designed: 5'-fragment as the miR-21 recognition unit, middle fragment as the miR-21 analogue amplification unit, and 3'-fragment as the 8-17 DNAzyme production unit. Triggered by miR-21 and accompanied by polymerase-nicking enzyme cascade, new miR-21 analogues autonomously generated for the successive re-triggering and cleavage process. Simultaneously, the 8-17 DNAzyme-contained sequence could be exponentially released and activated for the second cyclic cleavage toward a specific ribonucleotide (rA)-contained substrate, inducing a remarkably amplified generation of HRP-mimicking DNAzyme in the presence of hemin. Finally, the amperometric technique was used to record the catalytic reduction current of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The increase in the steady-state current was proportional with the increase of the miR-21 concentration from 1 aM to 100 pM. An ultra-low detection limit of 0.5 aM with an excellent selectivity for even discriminating differences between 1-base mismatched target and miR-21 was achieved. This simple and cost-effective 3TICA strategy is promising for the detection of any short oligonucleotides, simply by altering the target recognition unit in the template sequence.
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China; School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China.
Fang-Fang
FF
Cheng
College of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816, PR China; State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China.
Nan
N
Jiang
College of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816, PR China.
Xiaoyan
X
Li
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China.
Li
L
Zhang
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China.
Lihui
L
Hu
College of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816, PR China. Electronic address: chenxj_njut@126.com.
Xiaojun
X
Chen
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China. Electronic address: Jianglp@nju.edu.cn.
Li-Ping
LP
Jiang
Chemistry Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.
E S
ES
Abdel-Halim
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China.
Jun-Jie
JJ
Zhu
D001560:Benzidines / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D001940:Breast / Q000473:pathology; D001943:Breast Neoplasms / Q000097:blood* / Q000175:diagnosis; D021881:DNA, Catalytic / Q000737:chemistry; D055664:Electrochemical Techniques / Q000379:methods; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis / Q000097:blood*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods
D001560:Benzidines; D021881:DNA, Catalytic; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; C021758:3,3',5,5'-tetramethylbenzidine; D006861:Hydrogen Peroxide
Attomolar sensitivity; Electrochemical detection; Isothermal cascade reaction; Target-triggered triple amplification; microRNA-21