A core-shell-type fluorescent nanosphere possessing reactive poly(ethylene glycol) tethered chains on the surface for zeptomole detection of protein in time-resolved fluorometric immunoassay
A core-shell-type fluorescent nanosphere possessing reactive poly(ethylene glycol) tethered chains on the surface for zeptomole detection of protein in time-resolved fluorometric immunoassay
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DOI：
10.1021/ac034346e
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发表时间：
2003-11-15
影响因子：
7.4
通讯作者：
Kataoka, K
中科院分区：
文献类型：
--
作者：
Matsuya, T;Tashiro, S;Kataoka, K
为了提高生化分析的灵敏度并抑制非特异性结合，制备了一种与抗体共价结合的新型核壳型荧光纳米球（106.7 nm）。通过分散自由基聚合的苯乙烯的存在下，杂遥爪聚（乙二醇）（PEG）大分子单体，其中具有一个可聚合的乙烯基苄基基团在另一个链端和伯氨基基团，并使用作为表面活性剂的核-壳型纳米球的构建。所得纳米球在表面上具有PEG栓系链，其在PEG链的远端具有伯氨基（NH 2纳米球）。通过将荧光铕螯合物与β-二酮配体通过物理包埋方法结合在NH 2纳米球的核心中，构建荧光NH 2纳米球。然后使用杂交联剂将荧光NH 2纳米球上的伯氨基转化为马来酰亚胺基团。所得的纳米球在表面上具有马来酰亚胺基团（马来酰亚胺纳米球），在较温和的反应条件下，分子中具有SH基团的蛋白质可以定量地共价缀合到该纳米球上而不会使蛋白质变性。在用于甲胎蛋白（AFP）测定的模型夹心免疫测定中测试荧光纳米球的适用性。将抗人甲胎蛋白Fab'片段共价结合到马来酰亚胺纳米球（Fab'纳米球）上，用于甲胎蛋白（AFP）的固相时间分辨荧光免疫分析。甲胎蛋白的检出限（平均值+2标准差）为0.040 pg/mL或57.1 zmol（57.1 × 10（-21）mol，M-w，M-AFP = 70000）。在整个测定范围内的不精确度（浓度CV）为1.1%（100 pg/mL）- 17.1%（0.1 pg/mL），即使抗体与纳米球的这种缀合，非特异性结合几乎可以忽略不计（0.0008%），并且即使当将类似于1.9 × 10（9）个Fab'纳米球颗粒施加到微量滴定孔时也是如此。
To increase the sensitivity and to depress the nonspecific binding in biochemical assays, a new core-shell-type fluorescent nanosphere (106.7 nm) covalently conjugated with antibody was prepared. The core-shell-type nanosphere was constructed by dispersion radical polymerization of styrene in the presence of heterotelechelic poly(ethylene glycol) (PEG) macromonomer, which has a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end and used as well as a surfactant. The resulting nanosphere had PEG tethered chains on the surface, which possesses a primary amino group at the distal end of the PEG chain (NH2 nanosphere). The fluorescent NH2 nanosphere was constructed by incorporating fluorescent europium chelates with beta-diketonate ligands in the core of the NH2 nanosphere by means of a physical entrapment method. The primary amino groups on the fluorescent NH2 nanosphere were then converted to maleimide groups using a hetero cross-linker. The resulting nanosphere had maleimide groups on the surface (maleimide nanosphere), onto which proteins having SH group in the molecule could be covalently conjugated quantitatively without any denaturation of the proteins under the milder reaction condition. The applicability of the fluorescent nanosphere was tested in a model sandwich immunoassay for a-fetoprotein (AFP) determination. Anti-human AFP Fab' fragment was covalently conjugated onto the maleimide nanosphere (Fab' nanosphere), and it was used for the solid-phase time-resolved fluorometric immunoassay of AFP. The detection limit (mean + 2 SD) was 0.040 pg/mL or 57.1 zmol (57.1 X 10(-21) mol, M-w,M-AFP = 70 000) for AFP. The imprecision (concentration CV) over the whole assay range was 1.1% (100 pg/mL) - 17.1% (0.1 pg/mL), even though with this conjugation of antibody to the nanosphere, the nonspecific binding was practically negligible (0.0008%) and even when similar to1.9 x 10(9) particles of the Fab' nanosphere were applied to the microtitration well.