1. Where were the fluorescent signal molecules synthesized?
The fluorescent signal molecules BSA-FITC, PNA-RhB, and b-Lac-Cy5 were synthesized by Bersee Science and Technology Co., Ltd. and Fubo Biotechnology Co., Ltd. in Beijing, China. These molecules are essential for various research applications, including fluorescence microscopy and flow cytometry. The synthesis process involves the preparation of precursor molecules, followed by a series of chemical reactions to introduce the fluorescent groups. The resulting fluorescent signal molecules exhibit specific properties, such as excitation and emission wavelengths, which make them suitable for different experimental setups. The high-quality synthesis of these molecules ensures their reliability and reproducibility in research studies, contributing to the advancement of scientific knowledge in the field of chemical and biological research.
read more
2. What instruments were used for AuNPs absorption spectra?
The absorption spectra of AuNPs were performed on a Cary Series UV-vis spectrophotometer. Additionally, Fouriertransform infrared spectra were obtained using a Bruker Tensor 27 FTIR spectrometer. Excitation and emission spectra were acquired on a Cary Eclipse Fluorescence Spectrophotometer. The morphology of AuNPs-PEI was analyzed using a JEOL JEM-200CX transmission electron microscope. Mastersizer 2000 particle size analyzer and Zeta sizer were used for measuring average size and zeta potential, respectively. Varioskan Flash and NanoZoomer 2.0 RS were used for fluorescence intensity and HE staining images.
read more
3. How is AuNPs-PEI prepared from HAuCl4?
AuNPs-PEI is prepared by dissolving 10 mg PEI in 1 mL ultrapure water, then adding 125 uL 1% HAuCl4 * 3H2O and diluting it into 2.5 mL ultrapure water. Slowly adding 100 uL PEI and stirring for 8 hours at room temperature without light results in AuNPs-PEI. The solution is filtered and stored for further experiments. This method is based on previous studies and utilizes PEI as a reducing agent and stabilizer.
read more
4. What is the optimal binding ratio of AuNPs-PEI and FLPs in fluorescence titration experiments?
The optimal binding ratio of AuNPs-PEI and FLPs was determined through fluorescence titration experiments. AuNPs-PEI with different volumes was added into a single FLP (0.015 nM each) to measure the corresponding fluorescence intensity. The quenching efficiency was calculated using the formula: Quenching efficiency (%) = 100 x (F0 - Fn)/F0, where F0 represented the fluorescence intensity of FLPs without AuNPs-PEI, and Fn was the fluorescence intensity after adding various volumes of AuNPs-PEI to FLPs. Nonlinear least-squares curve fitting analysis was used to calculate the binding constant (Ka) via a 1:1 binding model. The binding affinity of AuNPs-PEI to different FLPs was found to be FLP dependent, with the order of BSA-FITC (1.41 x 10 8 M -1 ) > b-Lac-Cy5 (7.43 x 10 7 M -1 ) > PNA-RhB (2.68 x 10 6 M -1 ). The fluorescence quenching reached saturation when the volume of AuNPs-PEI was 80 uL, and the fluorescence intensity of the AuNPs-PEI/FLPs sensor was stable in the pH range of 4.5-8.5 and unaffected by the presence of inorganic ions in urine.
read more