Mechanism of surface plasmon resonance sensing by indirect competitive inhibition immunoassay using Au nanoparticle labeled antibody.
Mechanism of surface plasmon resonance sensing by indirect competitive inhibition immunoassay using Au nanoparticle labeled antibody.
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DOI：
10.1016/j.talanta.2017.05.003
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发表时间：
2017-09
期刊：
影响因子：
6.1
通讯作者：
D. C. Kabiraz;K. Morita;Kazuhira Sakamoto;Toshikazu Kawaguchi
中科院分区：
文献类型：
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作者：
D. C. Kabiraz;K. Morita;Kazuhira Sakamoto;Toshikazu Kawaguchi
我们研究了表面等离子体共振（SPR）生物传感器的使用，使用了一种标记有au纳米粒子（Ab- aunp偶联物）的抗体（Ab）。由于盐酸克仑特罗是一种小分子物质，故采用间接竞争抑制免疫分析法。使用Ab- aunp偶联物进行SPR免疫分析，检测限极低（LOD），为0.05 ppt (0.05 pg mL−1)，比未标记的Ab低40倍。为了确定间接竞争抑制免疫分析LOD的关键因素，评估了表面免疫反应（K1）和预混溶液（αK2）的亲和常数。我们发现，由于Ab的AuNP标记导致介电常数的变化对亲和常数没有影响，因为所有的放大幅度项在方程中都被抵消了。因此，确定k1和α k2值分别为3.0×1011M−1和2.9×1012M−1，分别比未标记的Ab高3和4个数量级。k1和α k2的LOD模拟图显示，ak1比α k2低1个数量级产生ppt级LOD。由于亲和常数由反应物和生成物的摩尔浓度决定，因此Ab或Ab- aunp共轭物的摩尔质量在含有1ppm （1 μg mL−1）的样品溶液中对该常数有很大影响。因此，摩尔质量调整可用于调整间接竞争抑制免疫测定中的LOD，以满足实际应用的需要。
We investigated the use of a surface plasmon resonance (SPR) biosensor using an antibody (Ab) labeled with Au-nanoparticle (Ab-AuNP conjugate). As clenbuterol is a small molecule, an indirect competitive inhibition immunoassay was used. The SPR immunoassay using Ab-AuNP conjugate had an extremely low limit of the detection (LOD) with a magnitude of 0.05 ppt (0.05 pg mL−1), which was 40 times lower than that of unlabeled Ab. To identify the key factor in determining the LOD of the indirect competitive inhibition immunoassay, affinity constants of the surface immunoreaction (K1) and of the premixed solution (αK2) were evaluated. We found that the dielectric constant change due to AuNP labeling of Ab did not affect on the affinity constants, because all the amplification magnitude terms canceled out in the equations. Thus, theK1and αK2values were determined to 3.0×1011M−1and 2.9×1012M−1, respectively, which were three and four orders of magnitude higher, respectively, than those of unlabeled Ab. The simulation plot of LOD with respect toK1and αK2showed that aK1one order of magnitude lower than αK2produced a ppt level LOD. Because the affinity constants are determined by the molar concentrations of reactant and product, the molar mass of the Ab or Ab-AuNP conjugate in the sample solution containing 1 ppm (1 μg mL−1) highly affects the constants. Consequently, molar mass adjustment can be used to adjust the LOD in an indirect competitive inhibition immunoassay as needed for a practical application.