Figure 1.
Spectral properties of EDANS-Dabcyl pair [45] and the flowchart of the experimental design.
(A) EDANS-Dabcyl is a widely used donor-quencher pair. The optimal absorbance and emission wavelengths of EDANS are λabs = 336 nm and λem = 490 nm respectively, and for Dabcyl, the maximum absorbance wavelength is λabs = 472 nm, which, to a large extent, overlap with the emission spectra of EDANS. When they are in a close proximity (10–100 Å), the energy emitted from EDANS will be quenched by Dabcyl, resulting in low or no fluorescence; when they are separated upon substrate cleavage, for example in this design, the fluorescence will increase. Hence from the fluorescence intensity change, the enzyme could be detected continuously and directly. (B) Based on the principle of FRET and our previous study, we chose the optical LC/B cleavage length of VAMP2 (63–85) as the linker between EDANS-Dabcyl.
Figure 2.
The specificity of the synthesized FVP-B.
LCs (200 nM each) were mixed with 8.4 µM FVP-B in reaction buffer to a final volume of 500 µl, incubated at 37°C for 1 h, fluorescent intensity was measured by a Fluorescence Spectrometer in quartz cuvette. The data obtained were processed with GraphPad Prism.
Figure 3.
LC/MS analysis of specific cleavage of FVP-B by LC/B.
Mixture sample of FVP-B (8.4 µM) and LC/B (200 nM) from Figure 2 was analysed by LC/MS to detect the full length FVP-B (A), CT-product (B) and NT-product (C).
Figure 4.
The feasibility of the synthesized FVP-B and our developed assay.
(A) 228 nM purified LC/B was mixed with 8.4 µM FVP-B in reaction buffer to a final volume of 500 µl. Reaction was incubated at 37°C and the fluorescent intensity was measured in quartz cuvette every 30 min. The total duration of measurement was 8 h. The data were processed by Origin85. (B) The fluorescent intensity peak at 502 nm was selected to represent the trend of the whole data. The data were processed with Excel.
Figure 5.
Optimization of the developed assay system.
To optimize the assay and test the sensitivity of our detection assay, ten-fold dilution of LC/B was performed with the concentration of FVP-B fixed at 8.4 µM (A); five-fold dilution of FVP-B, with the LC/B concentration fixed at 22.8 nM (B); the fluorescent intensity change at 502 nm of further diluted LC/B to 22.8 pM with 1.68 µM FVP-B and extended incubation time (C); and a spectrum representative of 22.8 pM LC/B incubated with 1.68 µM FVP-B for extended time span (D). Viewing from the data, the detection sensitivity of the developed assay is about 22.8 nM of LC/B within 30 min, but the sensitivity can be improved to 22.8 pM of LC/B with 2 h incubation. The data were processed by Origin85 and Excel.
Figure 6.
Limit of detection of the developed LC/B detection system.
Serial LC/B concentrations were incubated with 8.4 µM FVP-B in 500 µl reaction volume for 1 h incubation at 37°C. The fluorescence intensities were potted versus the concentration of LC/B. For each concentration, at least five replicates were carried out. LOD = 3*S/k (S means standard deviation of negative control, k means slope).
Figure 7.
The inhibitory effect of EDTA on LC/B.
228 nM of LC/B was mixed with 8.4 µM FVP-B with reaction buffer to a final volume of 500 µl, incubated at 37°C for 1 h, and then the fluorescent intensity was measured by a Fluorescence Spectrometer in quartz cuvette (A); 100 µl reaction volume, which contained 228 nM LC/B with 8.4 µM FVP-B, were carried out in 96 well plate after 1 h incubation at 37°C (B). For simplicity, the fluorescent intensity peak at 502 nm was selected. The data obtained were generated from at least three times repeats, and then processed with Excel and GraphPad Prism, with the negative control data subtracted.