A spherical poly(acrylic acid) brush-enzyme block with high catalytic capacity for signal amplification in digital biological assays

Ultrasensitive determination of some ultra-low abundance biological molecules closely related to diseases is currently a wide concern and urgent issue to be addressed. Here, a spherical poly(acrylic acid)-alkaline phosphatase (SP-AKP) signal amplification block using spherical poly(acrylic acid) brush nanoparticles (SP) as the immobilized carriers was designed and synthesized optimally first. The results show that a single SP-AKP with high enzyme binding capacity and high catalytic ability (up to about 4800 effective free AKP per SP-AKP) has much greater fluorescence signal amplification ability than a single free AKP or SiO-COOH-AKP. Then, a droplet generation microfluidic chip was prepared successfully, and the SP-AKP was loaded and confined in a 14 pL droplet by adjusting its concentration to ensure at most one SP-AKP was encapsulated in each droplet according to Poisson's theory. Finally, the fluorescence signals produced by 4-methylumbelliferyl phosphate (4-MUP) catalyzed via SP-AKP within 6 min were sufficient to be detected by a fluorescence microscope. Thus, the digital signal distribution of "1/0" (signal/background) was obtained, making this SP-AKP signal amplification block a promising enzyme label for potential high sensitivity digital biological detection applications.