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Automatic and Integrated Micro-Enzyme Assay (AIμEA) Platform for Highly Sensitive Thrombin Analysis via an Engineered Fluorescence Protein-Functionalized Monolithic Capillary Column
journal contribution
posted on 2015-04-21, 00:00 authored by Lihua Lin, Shengquan Liu, Zhou Nie, Yingzhuang Chen, Chunyang Lei, Zhen Wang, Chao Yin, Huiping Hu, Yan Huang, Shouzhuo YaoNowadays, large-scale screening for
enzyme discovery, engineering,
and drug discovery processes require simple, fast, and sensitive enzyme
activity assay platforms with high integration and potential for high-throughput
detection. Herein, a novel automatic and integrated micro-enzyme assay
(AIμEA) platform was proposed based on a unique microreaction
system fabricated by a engineered green fluorescence protein (GFP)-functionalized
monolithic capillary column, with thrombin as an example. The recombinant
GFP probe was rationally engineered to possess a His-tag and a substrate
sequence of thrombin, which enable it to be immobilized on the monolith
via metal affinity binding, and to be released after thrombin digestion.
Combined with capillary electrophoresis-laser-induced fluorescence
(CE-LIF), all the procedures, including thrombin injection, online
enzymatic digestion in the microreaction system, and label-free detection
of the released GFP, were integrated in a single electrophoretic process.
By taking advantage of the ultrahigh loading capacity of the AIμEA
platform and the CE automatic programming setup, one microreaction
column was sufficient for many times digestion without replacement.
The novel microreaction system showed significantly enhanced catalytic
efficiency, about 30 fold higher than that of the equivalent bulk
reaction. Accordingly, the AIμEA platform was highly sensitive
with a limit of detection down to 1 pM of thrombin. Moreover, the
AIμEA platform was robust and reliable to detect thrombin in
human serum samples and its inhibition by hirudin. Hence, this AIμEA
platform exhibits great potential for high-throughput analysis in
future biological application, disease diagnostics, and drug screening.