Fluorescence in situ hybridization (FISH) is a highly specific technique for detecting pathogenic bacteria, which does not require cultivation and provides information on the abundance, morphology, and spatial localization of the pathogenic bacteria. However, traditional FISH has limited sensitivity and poor multiplexing capability, which hinders its wider application. This paper proposes a fingerprint FISH (FinFISH) strategy based on DNA self-assembly for the detection of multiple pathogenic bacteria. FinFISH uses respiratory pathogens as a representative model and combines three different fluorescent dyes for labeling, generating distinguishable fluorescent fingerprints for each species. In this work, FAM, Cy3, and Cy5 were selected as fluorescent reporters because they represent mature combinations of fluorescent dyes for multi-color imaging and combinatorial encoding, with minimal spectral overlap under standard fluorescence microscopy conditions. This strategy enables pathogen detection to exceed the limitations of the number of fluorescence channels and effectively overcome the throughput bottleneck of traditional imaging systems, while maintaining high scalability for further expansion. Additionally, the custom-designed closed chip is equipped with multi-channel reaction chambers, enhancing parallel sample processing and simplifying experimental operations. Experimental results show that FinFISH performs well in identifying pathogenic bacteria in simulated sputum and urine samples and is applicable to clinical samples. Moreover, FinFISH provides additional semi-quantitative insights into mixed infections. With the future integration of extended probe design and AI-assisted analysis, FinFISH is expected to drive clinical pathogenic bacteria diagnosis, microbial co-localization research, and spatial analysis of bacteria within tumors. This study was published in ACS Nano under the title "Fingerprinting Fluorescent In Situ Hybridization Enables Multiplexed Identification of Pathogenic Bacteria"
References: DOI: 10.1021/acsnano.5c1884