异硫氰酸荧光素及FITC抗体应用

公司新闻 2023年7月17日 489

异硫氰酸荧光素(FITC)标记是一种常见的技术，具有广泛的应用范围，因为它与胺反应迅速，并且由于其高量子效率。由于其高分子吸收率，使用FITC标签比传统的比色标签和无线电标签更受欢迎，因为像FITC这样的荧光团明亮，更容易使用，并且不需要特殊的废物处理。FITC标记的蛋白质、底物、肽激素和抗体可以用作流式细胞术、酶动力学和免疫细胞化学的探针，也可以用于检测靶细胞表面的受体。
FITC的激发波长为494nm，发射波长为518nm，呈现绿色。最初，据报道FITC有两个异构体-荧光素5-异硫氰酸酯和荧光素6-异硫氰酸酯。已知存在两种异构体的产物(荧光素5(6)-异硫氰酸酯)。

Fluorescein isothiocyanate (FITC) labeling is a common technique with a wide range of applications because it reacts quickly with amines and due to its high quantum efficacy. Due to its high molecular absorptivity, using FITC labels is preferred over conventional colorimetric labels and radio labels because fluorophores like FITC are bright, easier to work with, and don’t require special waste handling. Proteins, substrates, peptide hormones, and antibodies labeled by FITC can be used as probes in flow cytometry, enzyme kinetics, and immunocytochemistry, as well as in the detection of receptors on the surface of the target cells.
The excitation and emission wavelengths of FITC are 494nm and 518 nm, respectively, which gives it a green color. Originally, FITC was reported to have two isomers – fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate. A product of both isomers is also known to exist (fluorescein 5(6)-isothiocyanate).

Scheme 1 a Preparation procedure of dual-codified gold nanoparticles with polyclonal antibody and dsDNAs-FITC. b A schematic illustration of the amplifying fluorescence immunoassay for detection of BPA

纳米颗粒(NPs)的表面修饰可以增强细胞和细胞内靶向。设计并合成了一种新型的聚胺修饰金纳米粒子(AuNPs)，该纳米粒子可以选择性地吸附在细胞膜上。制备了平均直径为4.0 nm的AuNPs，并用多胺(R-4C)进行酰胺化修饰。为了通过荧光成像检测细胞内NPs的分布，用异硫氰酸荧光素(FITC)功能化AuNP@MPA-R-4C。荧光标记的NPs AuNP@MPA-R-4C-FITC在几种细胞系中显示出最小的细胞毒性。激光共聚焦扫描显微镜和透射电镜均显示AuNP@MPA-R-4C-FITC分布在细胞膜上。与游离有机染料相比，修饰后的AuNPs在处理10分钟后，在细胞膜上的积累量显著增加。这些结果表明AuNP@MPA-R-4C-FITC可以作为靶向细胞膜的生物探针，用于各种生物学应用。

The surface modification of nanoparticles (NPs) can enhance cellular and intracellular targeting. A new type of polyamine-modified gold NPs (AuNPs) are designed and synthesized, which can be selectively absorbed onto the cell membrane. AuNPs with an average diameter of 4.0 nm were prepared and modified with polyamine (R-4C) through amidation. In order to detect the distribution of NPs within cells by fluorescence imaging, AuNP@MPA-R-4C was functionalized with fluorescein isothiocyanate (FITC). The fluorescence-labled NPs AuNP@MPA-R-4C-FITC demonstrated minimal cytotoxicity in several cell lines. Both confocal laser scanning microscopy and transmission electron microscopy demonstrated that AuNP@MPA-R-4C-FITC was distributed on the cell membrane. Compared with the free organic dye, the modified AuNPs showed significantly increased accumulation on the cell membrane after treatment for only 10 min. These results suggested that AuNP@MPA-R-4C-FITC can be used as a bioprobe targeting the cell membrane for various biological applications.

FITC分子可以很容易地通过它们的异硫氰酸基团附着在柠檬酸盐覆盖的aunp表面。FITC-AuNPs的荧光弱主要是由于FITC到AuNPs的有效荧光共振能量转移。发现2-ME的存在使FITC通过形成Au-S键从AuNPs表面去除，从而恢复FITC的荧光。相反，当2-ME在碱性条件下与ROS氧化时，生成的2-ME二硫化物无法去除NP表面的FITC。因此，在加入浓度增加的ROS后，FITC-AuNPs的弱荧光逐渐增强。离心去除AuNPs后，该方法对ROS的敏感性明显提高。由于葡萄糖氧化酶催化葡萄糖氧化产生葡萄糖酸和H2O2，因此该方法也用于葡萄糖的检测。在最佳条件下，H2O2、超氧阴离子、羟基自由基和葡萄糖的最低检测浓度分别为1 μM、0.6 μM、0.6 μM和1 μM。该方法已成功地应用于血清中葡萄糖的测定。

FITC molecules can be readily attached to the surface of citrate-capped AuNPs through their isothiocyanate group. FITC-AuNPs fluoresce weakly mainly due to the efficient fluorescence resonance energy transfer from FITC to AuNPs. It is found that the presence of 2-ME enables FITC to be removed from the surface of the AuNPs through the formation of Au–S bonds, thereby restoring the fluorescence of FITC. In contrast, when 2-ME is oxidized with ROS under alkaline condition, the generated 2-ME disulfide is unable to remove FITC from the NP surface. As a result, the weak fluorescence of FITC-AuNPs increased gradually after adding increasing concentrations of ROS. The sensitivity of this method toward ROS was significantly improved after removal of the AuNPs by centrifugation. Because the glucose oxidase-catalyzed oxidation of glucose yielded gluconic acid and H2O2, this method was also utilized to detect glucose. Under optimum conditions, the minimum detectable concentrations for H2O2, superoxide anion, hydroxyl radical, and glucose were found to be 1, 0.6, 0.6 and 1 μM, respectively. This method has been successfully applied to the determination of glucose in serum.

蛋白电晕定量，取10 uL AuNP溶液与40µL缓冲液和50µL KCN 100 mM混合，30 min后使AuNP完全溶解，通过荧光强度测定(λ激发= 480 nm， λ发射= 530 nm)定量蛋白含量。作为确认AuNP数的另一种方法，从同一样品中稀释10µL进行紫外可见分光光度测定。For protein corona quantification, 10 uL of AuNP solution was mixed with 40 µL of buffer and 50 µL KCN 100 mM. After 30 min, the complete dissolution of AuNPs was achieved, and the protein content was quantified by fluorescence intensity measurement (λexcitation = 480 nm and λemission = 530 nm). As an additional method to confirm the AuNP number, a UV-Vis spectrophotometric determination was performed by diluting 10 µL from the same sample.

荧光化学物质(如异硫氰酸荧光素)被偶联(附着)到抗体的恒定区。当标记抗体与测试样品孵育时，如果存在，抗体将与抗原结合。未结合的抗体被冲走。在荧光显微镜下，抗原存在的区域显示为荧光苹果绿。如果没有特定的抗原，就不会有荧光。Fluorescent chemicals (e.g fluorescein isothiocyanate) are conjugated (attached) to the constant region of antibodies. When labeled antibodies are incubated with a test sample, the antibody will bind to an antigen, if present.  Unbound antibodies are washed away. Areas, where antigens are present, are visualized as fluorescent-apple-green using a fluorescence microscope. If specific antigens are absent there will be no fluorescence

可视化读数示意图。生物素fitc标记的RAA扩增物首先与胶体金标记的抗fitc抗体结合。随着偶联物的流动，链亲和素可以在检测线上捕获偶联物，而抗小鼠抗体可以在对照线上捕获游离多余的胶体金标记的抗fitc抗体。在测试线和控制线上的视觉带表示正读数，而只有控制线上的单个带表示负读数。Schematic of LFA visual readout. Biotin-FITC-labeled RAA amplificons firstly conjugate with colloidal gold labeled anti-FITC antibodies. As the conjugate flowing, streptavidins can capture the conjugate at test line, while anti-mouse antibodies can capture free excess colloidal gold labeled anti-FITC antibodies at control line. Visual bands at both test line and control lines indicates a positive readout while only a single band at control line indicates a negative readout.

异硫氰酸荧光素及抗FITC抗体应用广泛，尤其是FITC-抗FITC系统，在磁微粒化学发光，免疫层析，核酸POCT中逐步得到应用，增强信号放大，性能稳定