Cyanine 5 Tyramide

Application Method of Cyanine 5 Tyramide for Detecting Hydrogen Peroxide and Glucose^[1]

This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.

1. Reagent Preparation: Cyanine 5 Tyramide (900nM); Horseradish peroxidase (HRP, Sigma-Aldrich); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for conjugation; Glucose oxidase (GOx, BBI); Tris buffer (100mM, pH=7.5); Hydrogen peroxide (H₂O₂) standard solution; Glucose standard solution; Bovine serum albumin (BSA) for blocking.

2. Preparation of QD-HRP Conjugates: Mix 24pmol of 605nm QDs with 480pmol of HRP (molar ratio 1:20) in 30μL of phosphate buffer (20mM, pH=6.0). Add 48nmol of EDC and 48nmol of NHS, and incubate at room temperature for 2 hours. Then, add BSA to a final concentration of 1mg/mL and continue incubation for another hour for blocking. Purify the QD-HRP conjugates using an ultrafiltration membrane (100kDa molecular weight cutoff). The final product is stored in borate buffer (4°C). Characterize the conjugates by capillary electrophoresis (CE).

3. Hydrogen Peroxide Detection: The reaction system contains 20nM QD-HRP conjugates and 900nM Cyanine 5 Tyramide in 100mM Tris buffer (pH=7.5). Add 20μL of H₂O₂ solution at various concentrations (0-4.0μM) to 180μL of the reaction mixture and incubate at room temperature for 10 minutes. Measure the fluorescence spectrum using a fluorescence spectrophotometer with an excitation wavelength of 488nm, recording the fluorescence intensity at 605nm (QDs emission) and 670nm (Cy5 emission). The Cy5 fluorescence intensity shows a linear relationship with H₂O₂ concentration in the range of 10-100nM, with a detection limit of 10nM.

4. Glucose Detection: The reaction system contains 20nM QD-HRP conjugates, 900nM Cyanine 5 Tyramide, and 5U/mL glucose oxidase in 100mM Tris buffer (pH=7.5). Add 20μL of glucose solution at various concentrations (0-20.0μM) to 180μL of the reaction mixture and incubate at 37°C for 5 minutes. Measure the fluorescence spectrum. The Cy5 fluorescence intensity shows a linear relationship with glucose concentration in the range of 50-500nM, with a detection limit of 50nM.

Precautions:

(1) A reaction time of 10 minutes is sufficient. Detection should be performed promptly after incubation.

(2) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:
[1] Huang X, Wang J, Liu H, et al. Quantum dot-based FRET for sensitive determination of hydrogen peroxide and glucose using tyramide reaction. Talanta. 2013 Mar 15;106:79-84.