A Preliminary Study On The Spherical Polyelectrolyte Brushes-enzyme Signal Amplification System Based On Microdroplet And Its Application In Digital ELISA

Spherical polyelectrolyte brushes(SPBs)is a new kind of material formed when one end of linear polyelectrolyte chains densely attached to spherical carriers such as nanoparticles.It had high protein binding capacity and maintained good biological activity due to the unique three-dimensional space structure formed by soft high-density polyelectrolyte chains grafted on surface.Therefore,spherical polyelectrolyte brushes can be used as biomacromolecular carrier or signal marker material with great application potential.This thesis built a spherical poly(acrylic acid)-enzyme(SP-AKP)signal amplifier unit,which has the characteristics of uniformity,stability and good colloidal dispersion,based on spherical poly(acrylic acid)brushes(SPAABs).Through optimizing the reaction conditions of enzyme conjugation,we obtained the SP-AKP with high enzyme binding capacity and high catalytic ability,and then characterized by enzyme linked immunosorbent assay(ELISA)method.The results showed that the SP-AKP had great fluorescence signal amplification ability and high detection sensitivity.In addition,a microdroplet generation and detection platform suitable for the high-performance spherical poly(acrylic acid)-enzyme signal amplifier unit was proposed in this thesis to realize the single-molecule digital detection.Firstly,we designed and prepared microfluidic chips,optimized injection method and signal detection method,and obtained a large number of uniform stable microdroplets.Secondly,by adjusting the concentration of SP-AKP,each droplet could contain at most one SP-AKP.Finally,optical imaging was used to record the number of fluorescent droplets generated by single SP-AKP catalyzed fluorescence substrate and the number of empty droplets without SP-AKP,so as to realize the expression and detection of digital fluorescent signals.The detection limit of this digital detection method for SP-AKP was up to 200 aM,which was lower than that of conventional detection method,indicating that this SP-AKP signal amplifier unit proposed in this paper has the potential to be applied to digital ELISA detection,and this microdroplet based SP-AKP signal amplification system is expected to achieve ultrasensitive digital biological detection.