[In-site electrophoretic elution of excessive fluorescein isothiocyanate from fluorescent particles in gel for image analysis].

The sensitivity, accuracy, and efficiency of fluorescent particle detection can be improved by purifying the fluorescent-dye-labeled particles. In this study, an in-site model of electrophoretic elution (EE) was developed for the facile and efficient removal of unconjugated fluorescent dyes after labeling reactions, thereby facilitating the sensitive fluorescent imaging of proteins captured by microbeads. First, bovine serum albumin (BSA) and magnetic beads (MBs) were chosen as the model protein and particles, respectively, and an MBs-BSA complex was synthesized by mixing the beads with the BSA solution. Second, excessive fluorescein isothiocyanate (FITC) was added to the EP tube with MBs-BSA suspension for the fluorescent labeling of BSA, and a labeled compound was obtained after 8-h incubation in the dark at 4 ℃. The unpurified MBs-BSA was obtained by removing the supernatant, leaving 5 μL of the residual solution in the EP tube. The obtained MBs-BSA solution was added to a 50-μL phosphate buffer solution (PBST, containing 0.01% Triton X-100, pH 7.4). Third, gel suspension was prepared by mixing the MBs-BSA solution with the low-gelling-temperature agarose gel (10 g/L) and filled into an electrophoresis channel. To demonstrate the high efficiency of the in-site model of EE for removing excessive FITC, a 10-mm hydrogel segment was prepared using MBs-BSA sandwiched between two blank hydrogels and filled into a 50-mm-long electrophoresis tube (outer diameter: 5 mm; inner diameter: 3 mm) for the EE. Subsequently, the filled channel was set in an electrophoresis device to construct the in-site EE model. The particle size of the MBs was larger than the pore size of the gel, and the fluorescent beads were physically immobilized in the gel while the excessive FITC was removed from the channel by electrophoresis. Before an EE run, the original fluorescence image of the target gel was captured using a CCD camera. After the 30-min EE (50 V, 6 mA, pH 7.4 PBS), the fluorescence image was also recorded by the CCD camera. The fluorescent images were converted to a grayscale intensity map. To simplify the calculation, a simple fluorescent image analysis method was developed. The side view of the grayscale intensity map is a two-dimensional plot of peaks. Each peak indicates a fluorescent spot at a given position along the length of the channel when the distribution density of the particles is low, and the peak value is the grayscale intensity of the fluorescent spot. The statistical peak numbers and values can be used to approximate fluorescent spots on the image. After image processing and calculations, 27.8% of the average grayscale intensity of the fluorescent spot was retained, comparing the average gray value of the bright spot before and after EE, and 97.6% of excessive FITC in the channel was cleared, obtained by calculating the decreased background fluorescence grayscale intensity after EE. The particle-to-background signal ratio (P/B ratio, PBr) increased from 1.08 to 12.2 after EE with an exposure time of 1.35 s. In addition, different exposure times were explored during the fluorescence detection. Increasing the exposure time from 1.35 to 2.35 s enhanced PBr from 12.2 to 15.5, which could effectively increase the signal-to-noise ratio. An appropriate increase in exposure time also allowed the detection of many weak fluorescent particles that were previously undetectable, indicating increased sensitivity of the fluorescence detection. The EE model has the following advantages: (i) increase in specificity by eluting FITC absorbed to the surface of beads; (ii) high efficiency in the removal of free FITC with more than 97% clearance; (iii) rapid decrease in noise in the mass hydrogel (within 30 min). This method can be used in beads/spots-based immunoassay in gel, immuno-electrophoresis, and fluorescent staining of protein/nucleic acid bands in gel electrophoresis.