2. Materials and Method
2.1Preparation and influencing factors of carbon quantum dot-biomolecular conjugate
(1) Preparation of conjugates [27]: 1 mL of water-soluble CQD solution was taken, EDC and NHS were added sequentially to activate quantum dots, and finally biomolecules were added to form a stable covalent conjugate. The resulting product was stored at 4 ℃ for testing.
(2) Influencing factors [28, 29]: The effects of EDC amount, NHS amount, carbon quantum dot-biomolecular ratio, reaction pH, and reaction time on the coupling reaction were investigated in a certain range. The best reaction conditions were determined by comparing and screening the fluorescence intensity.
a) Selection of coupling activator
Different concentrations of N-hydroxysuccinimide (NHS) were used as the second activator. Stable intermediates are formed with carboxylated quantum dots, which were then combined with biomolecules for coupling. The optimal concentration was determined by fluorescence intensity.
b) Influence of concentration ratio on coupler preparation
Based on the pre-experiment, the amount of EDC, the amount of NHS and the pH were kept unchanged, and the ratio of carbon quantum dots to biomolecules was adjusted. The optimal concentration ratio was determined by the fluorescence intensity of the conjugate.
c) Effect of reaction time on coupler preparation
The quantum dots were activated at different times (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h) and the optimal activation time was determined by the fluorescence intensity of the quantum dots.
d) Effect of reaction pH
The conjugates were prepared at pH=5, 6, 7, 8 and 9, and the optimal pH value was determined by the fluorescence intensity of the conjugates.