title: Steric-dependent label-free and washing-free enzyme amplified protein detection with dual-functional synthetic probes.
abstract: Enzyme-catalyzed signal amplification with an antibody-enzyme conjugate is commonly employed in many bioanalytical methods to increase assay sensitivity. However, covalent labeling of the enzyme to the antibody, laborious operating procedures, and extensive washing steps are necessary for protein recognition and signal amplification. Herein, we describe a novel label-free and washing-free enzyme-amplified protein detection method by using dual-functional synthetic molecules to impose steric effects upon protein binding. In our approach, protein recognition and signal amplification are modulated by a simple dual-functional synthetic probe which consists of a protein ligand and an inhibitor. In the absence of the target protein, the inhibitor from the dual-functional probe would inhibit the enzyme activity. In contrast, binding of the target protein to the ligand perturbs this enzyme-inhibitor affinity due to the generation of steric effects caused by the close proximity between the target protein and the enzyme, thereby activating the enzyme to initiate signal amplification. With this strategy, the fluorescence signal can be amplified to as high as 70-fold. The generality and versatility of this strategy are demonstrated by the rapid, selective, and sensitive detection of four different proteins, avidin, O6-methylguanine DNA methyltransferase (MGMT), SNAP-tag, and lactoferrin, with four different probes.
authors: †Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC).
authors: Chia-Wen
authors: CW
authors: Wang
authors: †Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC).
authors: Wan-Ting
authors: WT
authors: Yu
authors: †Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC).
authors: Hsiu-Ping
authors: HP
authors: Lai
authors: †Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC).
authors: Bing-Yuan
authors: BY
authors: Lee
authors: †Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC).
authors: Ruo-Cing
authors: RC
authors: Gao
authors: †Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC).|‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC).
authors: Kui-Thong
authors: KT
authors: Tan
mesh_terms: D001360:Avidin / Q000032:analysis*; D024402:Carbonic Anhydrase II / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007781:Lactoferrin / Q000032:analysis*; D008024:Ligands; D015394:Molecular Structure; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D001360:Avidin; D019853:O(6)-Methylguanine-DNA Methyltransferase; D007781:Lactoferrin; D024402:Carbonic Anhydrase II