Chemical redox modulated switch-on fluorescence of carbon dots for probing alkaline phosphatase and its application in immunoassay

Early diagnosis is an important requirement for the early treatment of diseases and reliable detection methods for biomarkers are indispensable tools for early diagnosis. However, recent innovations in analytical technology are rarely employed widely due to multiple factors including the requirement of specialized equipment and infrastructures. Hence, much attention has been focused on exploring universal nanoprobes for routine practices. Here, we report a chemical redox modulated switch-on fluorescence strategy based on versatile carbon dots (CDs) for probing alkaline phosphatase (ALP) and human IgG (a model target). The fluorescence of the CDs is initially quenched by Fe(III) and then restored by ascorbic acid, which is generated from the hydrolysis of ascorbic acid 2-phosphate (AAP) by the catalysis of ALP. Based on that, a simple “switch-on” fluorescence assay for ALP was designed and leads to a sensitivity of as low as 0.8 U L−1. The proposed assay is applied in ALP sensing in human serum samples. Additionally, the application of the switch-on fluorescence strategy is successfully expanded to the immunoassay of human IgG in serum from healthy adults and patients with systemic lupus erythematosus. The developed sensing system could be devised for various target sensors and has promising potential in the field of biological analysis and clinical diagnostics.

3. Fig. S3 Fluorescent emission spectra of the CDs at different excitation wavelengths ranging from 290 nm to 380 nm with increments of 10 nm. 4. Fig. S4 Fluorescence response of CDs in the presence of different metal ions (A) and small compounds (B).(The concentration of metal ions and compounds were 100 µM) 5. Table S1 Fluorescence lifetimes obtained with three-exponential fit of the fluorescence decay curves of the CDs alone, CDs/Fe(III), and CDs/Fe(III)/AA, respectively.6. Fig. S5 (A  Wavelength(nm) Where τ 1, τ 2, τ 3 are the three components of the lifetimes, A is the background offset (a fitting parameter), and B1, B2, B3 are pre-exponential functions which are related to the emission intensity.The average lifetime τ avg can be calibrated according to Equation (2): ) Fluorescence spectra of CDs in the presence of 300 µM Fe(III) and different concentration of AA from 0 to 600 µM.(B) Relationship between fluorescence intensity and the concentration of AA. 7. Fig. S6 (A) The changes of fluorescence spectra of CDs in the presence of different composition: (a) mere CDs ; (b) CDs + Fe(III); (c) CDs + Fe(III) + AAP; (d) CDs + Fe(III) + AAP + ALP.(B) The change of fluorescence spectra of CDs in the presence of Fe(III) and ALP. 8. Fig. S7 Optimization of the experimental conditions for sensing ALP. 9. Table S2 Comparison of other CDs-based sensors for the determination of ALP. 10.Table S3 Determination of ALP in human serum samples.11.Fig. S8 Selectivity of the fluorescence immunoassay to human IgG (500 ng/mL) compared with other interfering proteins at the concentration of 500 ng/mL.S-3

Fig. S3
Fig. S3Fluorescent emission spectra of the CDs at different excitation wavelengths ranging from 290 nm to 380nm with increments of 10 nm.

Fig. S4
Fig. S4 Fluorescence response of CDs in the presence of different metal ions (A) and small compounds (B).(The concentration of metal ions and compounds were 100 µM)

Fig. S5 (
Fig. S5 (A) Fluorescence spectra of CDs in the presence of 300 µM Fe(III) and different concentration of AA from 0 to 600 µM.(B) Relationship between fluorescence intensity and the concentration of AA.

Fig. S6 (
Fig. S6 (A) The changes of fluorescence spectra of CDs in the presence of different composition: (a) mere CDs ; (b) CDs + Fe(III); (c) CDs + Fe(III) + AAP; (d) CDs + Fe(III) + AAP + ALP.All of the CDs concentrations were 1 µL/mL; Fe(III) concentrations were 300 µM in (a), (b) and (c); AAP concentrations were 50 mM in (c) and (d); ALP concentration was 1000 U/L in (d).Excitation: 320 nm and emission: 412 nm.(B) The change of fluorescence spectra of CDs in the presence of Fe(III) and ALP.
Fig. S8 Selectivity of the fluorescence immunoassay to human IgG (500 ng/mL) compared with other interfering proteins at the concentration of 500 ng/mL.

Table S1
Fluorescence lifetimes obtained with three-exponential fit of the fluorescence decay curves of the CDs alone, CDs/Fe(III), and CDs/Fe(III)/AA, respectively.
Note: The fluorescence lifetimes of the CDs under different conditions were measured.The fluorescence decays could be fit to the three-exponential Equation (1):

Table S2
Comparison of other CDs-based sensors for the determination of ALP.

Table S3
Determination of ALP in human serum samples.Goat IgG Rabbit IgG Mouse IgG Human IgG 0