FRET-BASED LACTATE NANOSENSOR COMPRISING BACTERIAL LLDR FOR QUANTIFICATION OF LACTATE AND METHODS FOR QUANTIFYING METABOLIC RATES AND LACTATE TRANSPORT

摘要

The rates of lactate production and lactate consumption are importantparametersof cell metabolism, with relevance for hypoxia/ischemia, cancer, diabetes andother pathological conditions. However, there are no available methods tomeasurethe rates of lactate production and consumption in single cells. A nanosensorfordetecting and quantifying lactate in different types of samples, such astissues,intra-cellular and subcellular compartments, with high spatial and temporalresolution is disclosed. The nanosensor is a FRET-based lactate nanosensorcomprising a bacterial LldR transcription factor between donor and acceptorfluorescent protein moieties. Methods comprising use of the nanosensor forquantifying the activity of lactate transporters, rates of cellular lactateproductionand cellular lactate consumption, and rate of mitochondrial pyruvateconsumptionare also disclosed. Methods for quantifying the transformation in energymetabolism that characterizes cancer cells with single-cell resolution and fordetecting interference of candidate drugs with mitochondrial energetics areadditionally disclosed.