Rapid and colorimetric assay for detection of S. pneumoniae based on hydrogen peroxide release and analysis using color image processing

Abstract

Although Streptococcus pneumoniae (S. pneumoniae) is routinely identified using various clinical methods, most existing techniques are complicated, time-consuming, and require expensive equipment. Consequently, there is a critical need for rapid, sensitive, and economical detection methods. Herein, we report a rationally designed colorimetric assay incorporating biocompatible anthocyanins for the rapid detection of S. pneumoniae via the naked eye and image processing software. Unlike conventional anthocyanin-based assays that rely on pH-dependent color changes, our approach uses anthocyanins as oxidative degradation probes rather than pH indicators. The detection mechanism relies on the oxidative degradation of anthocyanins by hydrogen peroxide (H₂O₂), a key virulence factor secreted by S. pneumoniae, which induces a distinct color shift to gray. We systematically evaluated the performance of this assay in various matrices, including growth medium, saline solution, and artificial cerebrospinal fluid. Bacterial suspensions of 1.0 and 1.5 McFarland were colorimetrically detected within 90 and 30 minutes, respectively, through naked-eye observation and Delta-E analysis. This study demonstrates a highly stable, cost-effective, and easy-to-prepare diagnostic tool that offers significant advantages over traditional methods for point-of-care clinical applications.