High-throughput pyrosequencing, carried out in millions of picoliter-sized reactors on a fiber-optic slide, is known for its longer read length. However, both optical crosstalk (which reduces the signal-to-noise ratio of CCD images) and chemical retention adversely affect the accuracy of chemiluminescence determination, and ultimately decrease the read length and the accuracy of pyrosequencing results. In this study, both titanium and oxidized aluminum films were deposited on the side walls and upper faces of micro-reactor slides to enhance optical isolation; the films reduced the inter-well crosstalk by one order of magnitude. Subsequently, chemical retention was shown to be caused by the lower diffusion coefficient of the side walls of the picolitersized reactors because of surface roughness and random pores. Optically isolated fiber-optic slides over-coated with silicon oxide showed smoother surface morphology, resulting in little chemical retention; this was further confirmed with theoretical calculations. Picoliter-sized micro-reactors coated with titanium-silicon oxide films showed the least inter-well optical crosstalk and chemical retention; these properties are expected to greatly improve the high-throughput pyrosequencing performance.high throughput analysis, picoliter-sized micro-reactor, surface coating Citation:Han W J, Yuan L N, Wei Q Q, et al. Improved picoliter-sized micro-reactors for high-throughput biological analysis.