Hybridization chain reaction and DNAzyme-based dual signal amplification strategy for sensitive colorimetric sensing of acetylcholinesterase activity and inhibitor screening in rat blood

•A colorimetric biosensor for AChE assay and inhibitor screening is developed.•Cu2+-specific DNAzyme and HCR are used for signal amplification.•This method is highly sensitive with a detection limit of 5 μU mL−1 AChE.•This assay can eliminate interference from common biological species. We have constructed a novel colorimetric sensing platform for quantitative detection of acetylcholinesterase (AChE) activity and its inhibitor (donepezil) in rat blood, which integrates the signal amplification of DNAzyme and hybridization chain reaction (HCR) with the assembly of gold nanoparticles (AuNPs). Herein, in the presence of AChE and its substrate acetylthiocholine (ATCh), the sensing system exhibits a dramatic decrease in absorbance at 522 nm, where AChE hydrolyzes ATCh into thiocholine, the resulting thiols capture Cu2+ from DNAzyme, and then the substrate strand of DNAzyme acts as an initiator to trigger HCR process. The HCR products can induce the assembly of AuNPs via DNA hybridization, accompanying by a sharp color-change from red to blue. When donepezil is added, the enzymatic activity of AChE is suppressed, resulting in an increase in the absorbance at 522 nm. Under optimal conditions, the colorimetric sensing platform shows sensitive responses to AChE and donepezil in the range of 0.02–1.5 mU mL−1 and 0.5–100 nM, respectively. The detection limits of AChE and donepezil are as low as 5 μU mL−1 and 0.5 nM, respectively. Owing to high sensitivity of the proposed method, various nonspecific interactions can be eliminated with a sufficient dilution, indicating that our strategy has great potential for practical application in neurobiology and pharmacology fields.