Functionalized gold nanoclusters as fluorescent labels for immunoassays: Application to human serum immunoglobulin E determination
Functionalized gold nanoclusters as fluorescent labels for immunoassays: Application to human serum immunoglobulin E determination
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DOI：
10.1016/j.bios.2015.08.011
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发表时间：
2016-03-15
影响因子：
12.6
通讯作者：
Sanz-Medel，Alfredo
中科院分区：
文献类型：
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作者：
Cruz Alonso，Maria;Trapiella-Alfonso，Laura;Sanz-Medel，Alfredo
建立了一种以金纳米团簇（AuNCs）为荧光标记物的免疫荧光定量分析方法。水溶性金纳米碳使用硫辛酸合成，然后彻底表征。所得金纳米粒子的粒径为2.7±0.1 nm，最大荧光发射波长为710 nm。合成的AuNCs显示出非常好的稳定性的荧光信号与曝光和在中性和弱碱性介质。通过碳二亚胺反应进行这些AuNC与所需抗体的共价生物缀合。在适当优化这种生物缀合反应后，选择摩尔比1：3（抗体：AuNC）。生物共轭物保持了强烈的发光发射，稍微红移相比，自由AuNCs。两个典型的免疫分析配置，竞争性和夹心，进行了测定和他们的IgE测定性能进行了严格的比较。在完成不同的免疫测定步骤后，测量生物缀合物的荧光发射。虽然夹心形式提供了10 ng/mL的检测限（DL）和25 - 565 ng/mL IgE的线性范围，竞争形式显示DL为0.2 ng/mL，线性范围为0.3 - 7.1 ng/mL。通过成功地分析人血清中的IgE，并将结果与免疫荧光法的结果进行比较，评估了更灵敏的竞争性荧光免疫测定法的适用性。一个商业工具包。所提出的基于AuNCs的荧光分析方法的主要优点包括低DL和涉及很少试剂的简单免疫测定方案。
A quantitative immunoassay for the determination of immunoglobulin E (IgE) in human serum using gold nanoclusters (AuNCs) as fluorescent label was developed. Water soluble AuNCs were synthesized using lipoic acid and then thoroughly characterized. The obtained AuNCs have a particle size of 2.7±0.1 nm and maximum fluorescence emission at 710 nm. The synthesized AuNCs showed very good stability of the fluorescent signal with light exposure and at neutral and slightly basic media. A covalent bioconjugation of these AuNCs with the desired antibody was carried out by the carbodiimide reaction. After due optimization of such bioconjugation reaction, a molar ratio 1:3 (antibody:AuNCs) was selected. The bioconjugate maintained an intense luminescence emission, slightly red-shifted as compared to the free AuNCs.Two typical immunoassay configurations, competitive and sandwich, were assayed and their performance for IgE determination critically compared. After the different immunoassay steps were accomplished, the fluorescence emission of the bioconjugate was measured. While the sandwich format provided a detection limit (DL) of 10 ng/mL and a linear range between 25 and 565 ng/mL of IgE, the competitive format revealed a DL of 0.2 ng/mL with a linear range between 0.3 and 7.1 ng/mLThe applicability of the more sensitive competitive fluorescent immunoassay was assessed by successful analysis of the IgE in human serum and comparison of results with those from a commercial kit. The main advantages of the proposed AuNCs-based fluorimetric method include a low DL and a simple immunoassay protocol involving few reagents.