“Fluorescence-wavelength” label-free POCT tandem with “fluorescence-photothermal” nanobody-immunosensor for detecting BSA and β-lactoglobulin

Two carbon dots (CDs) (λEm = 525 nm, G-CDs and λEm = 640 nm, R-CDs) were synthesized from citric acid and urea. The bovine serum albumin (BSA) responsiveness of the R-CDs was used to develop a “fluorescence-wavelength” label-free point of care testing (POCT) for the detection of the milk quality marker BSA with the detection limit (LOD) of 4.89 μg/mL for fluorescence mode and 3.38 μg/mL for wavelength mode. In addition, R-CDs were found to have hydroxyl radical (·OH)-dependent fluorescence quenching properties, and a “fluorescence-photothermal” immunosensor based on nanobodies was constructed by introducing the fluorescence signal of R-CDs@BSA and the photothermal signal of oxTMB for the detection of β-lactoglobulin (β-LG) with the LOD of 0.034 ng/mL for fluorescence mode and 0.075 ng/mL for photothermal mode. The tandem detection of POCT and immunosensor enables the simultaneous and highly sensitive detection of BSA and β-LG after only simple dilution of less than 5 µL of sample.