Developing an ultra-reproducible and ultrasensitive label-free nanoassay for L-methionine quantification in biological samples toward application in homocystinuria diagnosis

A novel, rapid, selective, ultrasensitive, and ultra-reproducible spectrofluorometric nanoassay was designed for the L-methionine quantification. A carbon dot with a %QY as high as 95% was prepared using a solvent-free ultrafast method and used as the label-free nanoprobe. The nanoprobe was characterized for size, morphology, elemental composition, optical properties, and quantum efficiency. Afterward, the nanoprobe was utilized for quantification of L-methionine in biological samples, revealing an ultra-wide linear range of 0.0–140.0 nm with a LOD as low as 0.65 nM. Besides, the developed method showed ultra-reproducibility with an inter-week assay, of RSD = 2.7% and high repeatability with a RSD as low as 1.0%. The mechanistic studies proved the simultaneous formation of two complexes between CDs and L-methionine, resulting in high sensitivity, selectivity, and stability of the sensor. Moreover, a binding constant as high as 1.4 × 10⁺⁶ M⁻¹, number of binding sites of 1.5, and a ∆G of − 35.1 was obtained for interaction between CDs and L-methionine. Finally, the developed method was utilized for analysis of the methionine content of human serum samples for both healthy and patients with homocystinuria for proving its possible application in homocystinuria diagnosis, showing highly accurate and reproducible results. Consequently, the proposed nanoassay can be applied for clinical practical applications toward homocystinuria diagnosis.