Colocalization recognition-activated cascade signal amplification strategy for ultrasensitive detection of transcription factors.
Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10-13M. Moreover, NF-κB p50 can be detected in as low as 0.21ngμL-1HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases.
Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
Desong
D
Zhu
School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China.
Lei
L
Wang
Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
Xiaowen
X
Xu
Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China. Electronic address: wjiang@sdu.edu.cn.
Wei
W
Jiang
D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic
D016328:NF-kappa B; D014157:Transcription Factors; D004247:DNA
Cascade signal amplification; Colocalization recognition; False positives; Transcription factors; Ultrasensitive detection