凝胶中荧光颗粒原位电泳洗脱过量异硫氰酸荧光素用于图像分析

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

去除荧光标记后残余荧光染料可以提高荧光颗粒检测的灵敏度、准确度和效率。该文发展了一种原位电泳洗脱(electrophoretic elution,EE)模型,用于在荧光标记后快速去除多余的荧光探针,实现荧光颗粒的灵敏检测。将牛血清蛋白(BSA)和磁珠(MBs)作为模式蛋白和微颗粒,混合孵育获得MBs-BSA,用异硫氰酸荧光素(FITC)对MBs-BSA标记,得到MBs-BSA_(FITC)复合物。将含有多余FITC的MBs-BSA_(FITC)溶液与低凝聚温度琼脂糖凝胶溶液按1∶5的体积比混合,并将混合物凝胶和纯琼脂糖凝胶分段填充到电泳通道中。电泳过程中,利用颗粒尺寸与凝胶孔径的差异来保留MBs-BSA_(FITC),同时将游离的FITC洗脱。经过30 min的电泳洗脱,通道内多余的FITC清除率达到97.6%,同时目标颗粒荧光信号保留了27.8%。成像系统曝光时间为1.35 s时,电泳洗脱将颗粒与背景的荧光信号比(P/B ratio,PBr)从1.08增加到12.2。CCD相机的曝光时间增加到2.35 s,可以将PBr提高到15.5,可进一步实现对微弱荧光亮点的高灵敏检测。该模型有以下优点:(1)能对颗粒表面非特异性吸附的FITC实现有效洗脱,提高了检测的特异性;(2)能够将97%以上的游离FITC清除;(3)30 min内能够使凝胶内的背景荧光大幅降低,提高了PBr和检测灵敏度。因此,该方法具有在凝胶中进行基于磁珠/荧光颗粒点的免疫检测、在免疫电泳或凝胶电泳中对蛋白质/核酸条带进行荧光染色等领域的应用潜力。

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_(FITC) was obtained by removing the supernatant,leaving 5μL of the residual solution in the EP tube.The obtained MBs-BSA_(FITC) 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_(FITC) 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_(FITC) 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.