Dual-channel foldable microfluidic paper-based parallel enzymatic reaction systems for simultaneous visual colorimetric detecting acetylcholinesterase and α-glucosidase together with screening inhibitors

On-site multi-enzymes detection and inhibitors screening are still challenging and particularly urgent. Here, dual-channel foldable microfluidic paper (μPAD)-based parallel enzymatic reaction systems were fabricated for simultaneous visual colorimetric detecting acetylcholinesterase (AChE) and α-glucosidase (α-Glu) together with screening inhibitors. μPAD composing of two layers with dual channels was designed and prepared facilely by a permanent marker. AChE and α-Glu catalyze pre-loaded substrates into products (thiocholine (TCh), ascorbic acid (AA)) on the 1st layer. Parallelly, oxidase-like MnO2 nanosheets oxidize pre-dropped colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) on the 2nd layer. After simple folding μPAD, TCh and AA can reduce oxTMB to TMB, resulting in color fading. Obviously, bioenzyme and nanozyme reactions under different pH can simultaneously occur on the μPAD. Furthermore, colorimetric results can be easily captured and on-site quantitatively analyzed by smartphone. As a result, a convenient, efficient, sensitive, and specific platform has been constructed for simultaneous detecting AChE and α-Glu in human serum samples (recoveries, 92.1–105.0%; relative standard deviations, < 3.6%), and screening inhibitors from synthesized compounds and natural products. The developed platform provides constructive insights for exploitation of visual detecting strategies for simultaneous sensing muti-targets for point-of-care testing of disease and development of drugs.