Highly sensitive protein biosensor based on a conjugated polymer brush

Simple and sensitive detection of proteins is crucial in biological analysis and medical diagnosis. Conjugated polymers (CPs) with π-conjugated backbones were recognized as having excellent light-harvesting capability and high fluorescent quantum yield. They have been widely used as an energy donor to amplify fluorescence signal via high efficient Föster resonance energy transfer (FRET). In particular, conjugated polymer brush with high charge density provides more possibilities due to stronger electrostatic interactions with negatively charged biomolecules. Here, we developed a highly sensitive protein biosensor for thrombin detection based on a conjugated polymer brush (PFNI) and a fluorescein-labeled aptamer (FAM-apt15). PFNI is a water-soluble cationic polyfluorene derivate with extremely high charge density (78 positive charges per repeat unit). PFNI can attract negatively charged aptamer through strong electrostatic interactions. In this case, the energy donor (PFNI) and acceptor (FAM) are in a close proximity, which results in an efficient FRET process and a high FRET signal. However, when the FAM-apt15 combines with the target protein, a rigid and big-sized G-quadruplex/thrombin complex formed. Due to the steric hindrance from the densely brush of PFNI, the distance between the two fluorophores increased significantly, leading to an inefficient FRET process and a low FRET signal. The strategy exhibits excellent specificity and the limit of detection (LOD) for thrombin in buffer was estimated to be 0.05 nmol/L. It also works well in diluted serum and a LOD of 0.2 nmol/L can be obtained. Compared to the biosensors based on traditional linear conjugated polymers, the sensitivity was improved by one order of magnitude. In addition, our strategy also shows the merits of simple, label- free, and low-cost because labeled DNA is much more expensive than unlabeled one. Based on the specific binding of aptamer and protein, this novel method can be extended to a highly sensitive detection of more proteins.