| Popis: |
Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. Metabolite detection is of particular interest in microbial biofilms. Mass spectroscopy (MS) techniques have been applied to metabolite detection in biofilms, but the instrumentation is bulky and expensive. Additionally, MS techniques focus on probing biofilm top surfaces, which requires various forms of colony treatment such as liquid bridges, imprinting on a substrate, coating with an organic matrix, or incorporation of tags. Scanning electrochemical microscopy (SECM) can be used to study electrochemically active metabolites on the top surface of biofilms in a spatially resolved fashion, but without the ability to quantify concentration or to simultaneously detect multiple redox-active species. Here, we demonstrate spatially resolved detection and profiling of metabolites released from biofilm bottom surfaces by interfacing biofilms to a complementary metal-oxide-semiconductor (CMOS) integrated circuit (IC). The chip contains an array of working electrodes and parallel potentiostat channels. "Images" over a 3.25-mm-by-0.9-mm area can be captured with a spatial resolution of 750 μm limited currently by diffusion through the thin (1-mm) agar film interfacing the biofilm with the chip. Using this platform, we demonstrate that square wave voltammetry (SWV) can be used to detect, identify, and quantify (for concentrations as low as 2.6 μM) four distinct phenazines, redox-active metabolites produced by Pseudomonas aeruginosa PA14. We characterize phenazine production in both wild-type and mutant P. aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine gene expression. Key to the methods developed in this work is the exploitation of IC technology as a new tool for biology, enabling many measurement channels and electrodes to be fabricated within a very small area. |
