Prussian blue (PB) has unique advantages and is widely used in the field of colorimetric detection. On the one hand, PB can convert light energy into heat energy under near-infrared light (NIR) irradiation, which is an excellent photothermal transducer and suitable for photothermal therapy (PTT) in the biomedical field. On the other hand, PB has excellent peroxidase-like (POD) activity, which can catalyze the oxidation of the color developer 3,3',5,5'-tetramethylbenzidine (TMB) to produce the blue oxide oxTMB in the presence of H2O2.
Both TMB and H2O2. are low-cost and non-toxic, and this catalytic oxidation reaction can be carried out in aqueous solution and at room temperature, making it ideal for in vitro diagnostics. oxTMB has a large molar extinction coefficient, which greatly enhances the sensitivity of the test. In addition, modification of PB can increase its specific surface area and provide more active sites, enhancing its POD-like activity.
Prussian blue nanoparticles (PBNPs), which combine the properties of nanomaterials and PBs, are increasingly used in biomedical applications. Studies have shown that due to their porous structure and easy surface modification, PBNPs are able to carry drug molecules and precisely release drugs in response to stimuli from the external environment, improving therapeutic efficiency. In the field of medical imaging, PBNPs can be used as imaging contrast agents to enhance image display due to their strong absorption in the near-infrared (NIR) region, high photothermal conversion efficiency and paramagnetism, as well as nano-enzymatic properties, which can catalyze the removal of excess reactive oxygen species (ROS) in the body, which can be used for photodynamic therapy of tumors and the treatment of ROS-related diseases.
PB can be used to develop a nanocatalyst PB/PM/HRP/Apt with good peroxidase (POD) activity, which catalyzes the oxidation of the color developer TMB to the blue oxide oxTMB in the presence of H2O2.
Fig. 1 Preparation process of PB/PM/HRP/Apt.
Materials and Reagents
- PB solution (0.2 mg/mL)
- PBS buffer
- Protease inhibitors
- TMB
- H2O2
- Horseradish Peroxidase-Biotin (HRP-Bio) solution (1 mg/mL)
- PD-L1 aptamer solution (10 µmol/mL)
- N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) solution (0.5 mol/L)
- N-Hydroxysuccinimide (NHS) solution (0.5 mol/L)
- FITC-Avidin (FITC-Avi) solution (0.5 mg/mL)
Equipment
Centrifuge, Sonicator, -80℃ freezer, Shaking incubator, UV-Vis spectrophotometer
Preparation Methods
Platelet membrane (PM) acquisition
Platelet-rich plasma (PRP) is obtained by centrifuging 10 mL of whole blood for 10 minutes and separating the supernatant. After centrifugation of the PRP at 3000 rpm for 20 minutes, the precipitate is ished with PBS buffer and repeated centrifugation is performed to obtain platelets (PLT). PLT is frozen at -80°C and then thawed at room temperature and repeated 3 times. PM are obtained by centrifugation at 8000 rpm for 10 minutes. The PM is ished with PBS containing protease inhibitors and sonicated for 5 min. The PM is dispersed in 1 mL of PBS buffer and stored at -20℃.
Synthesis of PB/PM nanoparticles
The 120 μL PM solution is mixed with 100 μL PB (0.2 mg/mL) and sonicated for 35 min to obtain platelet membrane-coated Prussian blue nanoparticles (PB/PM). To maintain the activity of membrane proteins, ice is added during sonication. Finally, the freshly prepared PB/PM is placed in a PBS buffer overnight at 4°C. The PB/PM is centrifuged at 8000 rpm for 6 minutes, the supernatant is discarded, and the precipitate is dissolved in PBS solution. This process is repeated three times to remove excess cell membrane to obtain purified PB/PM, which is dispersed in 1 mL of PBS solution and stored frozen at 4°C. The purified PB/PM/HRP/A is then stored in a PBS buffer overnight at 8000 rpm for 6 minutes.
Synthesis of PB/PM/HRP/Apt
To modify the surface of PB/PM, 50 μL of HRP-Bio solution (1 mg/mL), 50 μL of PD-L1 aptamer solution (10 μmol/mL), 10 μL of EDC solution (0.5 mol/L), and 10 μL of NHS solution (0.5 mol/L) are mixed homogeneously, and then the carboxylic acid groups are activated by shaking for 1 hour at 4 ℃. The activated HRP-Bio and PD-L1 aptamers are transferred to a centrifuge tube containing 1 mL of PB/PM and mixed homogeneously, and shaken for 12 h at 4 ℃ to obtain PB/PM/HRP/Apt. Purified PB/PM/HRP/Apt is obtained by centrifugation and the supernatant is discarded. Subsequently, the product is mixed with 10 μL of FITC-Avi solution (0.5 mg/mL) and incubated for 1 h to obtain the FITC fluorescent group-modified PB/PM/HRP/Apt.
