| Conjugated polyelectrolytes (CPEs) are water-soluble conjugated polymers with ionic side groups. Due to the uniqueπ-π* conjugated electronic structures and the remarkable signal amplification of conjugated polymers (CPs), as well as the good water-solubility and electrostatic behavior of the traditional polyelectrolytes, CPEs exhibit great potential in making high-sensitive bio- and chemo-sensors. Therefore, the use of CPEs as either chemical or biological sensing elements has received intense interest in the assays of various bio-molecules, e.g., saccharides, proteins, enzymes and DNA. In this work, the main efforts are focused on the synthesis of novel CPEs and the construction of sensitive assays for enzyme activity, with the contents including:1) By using a facile synthetic route, an anionic CPE with sulfonate groups (PPESO3) has been prepared. And the same route has been further applied successfully to the synthesis of a carboxylate-substituted polyphenyleneethynylene (PPE), PPE-OBS. On this basis, by integrating the two synthetic strategies for conjugated PPE and dendritic PAMAM, a novel CPE that features multiple carboxylate groups, PPE-(COOK)4, has been designed and synthesized.2) The properties of PPE-OBS and PPE-(COOK)4 have been investigated in detail. It is found that PPE-OBS suffers from the unstable emission and low quantum yields due to the poor water-solubility, which can be greatly improved via the addition of surfactant (CTAB or PVP). By contrast, PPE-(COOK)4 is characterized by good water-solubility, stable emission and high quantum yields. This is mainly because of the solubilizing effects of multiple ionic side groups and the enhanced electrostatic repulsion between these heavily charged macromolecules, which can prevent PPE-(COOK)4 from aggregating in aqueous solution.3) A label-free ultra-sensitive and selective assay of protease has been developed based on the aggregation/de-aggregation (fluorescence turn-off/on) of PPESO3. By taking advantage of the cationic peptide-induced aggregation of PPESO3, followed by an efficient fluorescence turn-on once the peptide substrate is cleaved, the proposed sensor protocol does not require any labeled probe, and can be used to assay different proteases. It is firstly found that Args can effectively quench the fluorescence of PPESO3, however, any cleavage will result in a notable decrease or even a complete loss of its capacity to make PPESO3 aggregate (quenching), which guarantees the ultra-high sensitivity of trypsin assay. The limit of detection is shown to be 0.25 ng/mL (about 11 pM), which is superior to those of most previous methods by 1-2 orders or higher. In addition, both the HPLC and MS characterizations have revealed a threshold length of oligo-polyarginine (Arg4) below which the trypsin-catalyzed hydrolysis is greatly slowed down. This attribute allows the specific assay of chymotrypsin through carefully designing the appropriate peptide length and the cleavage site, providing a limit of detection as low as 6 pM. This study opens a good opportunity for low-cost monitoring of protease activity in an ultrasensitive and selective manner.4) A novel fluorescence turn-on assay for glutathione reductase (GR) based on PPE-(COOK)4/Cu2+ has successfully been developed. It has been demonstrated that PPE-(COOK)4 shows both high sensitivity (KSV=4.8×106 M-1) and selectivity toward Cu2+. Moreover, by taking advantage of the distinct fluorescence recovery of PPE-(COOK)4/Cu2+ caused by GSH and by GSSG, a novel fluorescence turn-on assay for GR activity has been developed. With a minimum detectable concentration of 0.2 mU/mL, the proposed GR assay is highly sensitive and robust as compared to most spectrophotometric and fluorescent methods in use now. Thus, it is suitable for monitoring GR activity and can be used in screening potential drugs based on the inhibition of GR.
|
PDF Full Text Request