Revolutionizing Gram-negative bacteria detection: FLIM and multicolor imaging based selective interaction study using colistin passivated carbon dots

The rising prevalence of antibiotic-resistant Gram-negative bacteria presents a major challenge for healthcare and requires the development of new and effective detection techniques. In this study, we present a novel and simple method for the selective detection of Gram-negative bacteria using multi-emissive colistin-passivated carbon dots (m-CCD) based on Fluorescence Lifetime (FLT) response and bioimaging. The m-CCD was synthesized using a one-step microwave-assisted hydrothermal process, and its interaction with Gram-negative bacteria was studied using Fluorescence Lifetime Imaging Microscopy (FLIM) and automated Confocal Laser Scanning Microscopy (CLSM). The investigations revealed a notable reduction in the FLT of m-CCD-treated Gram-negative bacteria (average lifetime of m-CCD, (tau(avg)) (m-CCD) is 3.91 ns and m-CCD treated Gram-negative bacteria, (tau(avg)) (m-CCD-bac) is 1.57 ns), demonstrating the selective interaction between them. Furthermore, the CLSM data revealed high specificity of m-CCD for Gram-negative bacteria, even in polymicrobial samples. Importantly, we also demonstrate that this method can quantify Escherichia coli (E. coli) cells with two linear ranges such as 3.40 x 10(5) - 9.80 x 10(5) CFU mL(-1) and 6.90 x 10(7)-4.14 x 10(8) CFU mL(-1) in standard culture and real samples such as tap water and human urine with a limit of detection similar to 3.68-4.89 x 10(4) CFU mL(-1). This straightforward method has great potential for the rapid and selective detection of Gram-negative bacteria, paving the way for early diagnosis and treatment of bacterial infections.