Ratiometric fluorescent biosensors for quantitative lactic acid detection using CdTe@CdS quantum dots and lactate oxidase
Giuliana Grasso, Riccardo Scarfiello, Francesco Colella, Valentina Onesto, Stefania Forciniti, Giuseppe Gigli, Luigi Carbone, Loretta L. del Mercato
Abstract
• CdTe@CdS quantum dots and lactate oxidase allow H₂O₂-mediated fluorescence quenching. • Dual green/red quantum dot variations enhance versatility and stability in lactate sensing. • Biosensor offers rapid detection with limits as low as 0.0066 mM for physiological settings. Lactic acid plays a crucial role in various physiological processes, particularly in cellular metabolism and muscle activity, as it is a key marker of anaerobic respiration and metabolic stress. In this study, we present a ratiometric fluorescent biosensor for lactic acid detection, utilizing a fluorescence quenching mechanism. The sensor comprises a hydrogen peroxide (H 2 O 2 )-sensing unit, based on photoluminescent core-shell cadmium telluride@cadmium sulfide quantum dots (CdTe@CdS QDs), along with a reference probe covalently bound to silica microparticle (SiO 2 MPs), which acts as the substrate. Lactate oxidase (LOx) is immobilized on the microparticle surface, where it catalyzes the aerobic oxidation of l -lactate into pyruvate, generating H 2 O 2 in the process. The increasing concentrations of lactate (0–30 mM) result in proportional quenching of the CdTe@CdS QDs’ photoluminescence due to H 2 O 2 , while the reference fluorescence emission remains stable. A lactate calibration curve has been determined using confocal laser scanning microscopy (CLSM), enabling quantitative evaluation of the sensor's ratiometric response and colorimetric shifts via image analysis. To demonstrate the versatility of this approach, we engineered two variations of the microsensor with distinct ratiometric setups, one using green- and the other red-emitting CdTe@CdS QDs, paired with either rhodamine isothiocyanate (RBITC) or 7-(diethylamino)coumarin-3-carboxylic acid (7ACC1) as the reference dyes, both co-immobilized with LOx enzyme on the microparticle surface. These innovative microsensors offer a simple yet effective tool for the quantitative detection of lactic acid, leveraging its H 2 O 2 -sensing capability of CdTe@CdS QDs in combination with to LOx enzyme activity.