Järås Kerstin, Tajudin Asilah Ahmad, Ressine Anton, Soukka Tero, Marko-Varga György, Bjartell Anders, Malm Johan, Laurell Thomas, Lilja Hans
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, Lund, Sweden.
J Proteome Res. 2008 Mar;7(3):1308-14. doi: 10.1021/pr700591j. Epub 2008 Feb 8.
To improve the sensitivity of antibody microarray assays, we developed ENSAM (Europium Nanoparticles for Signal enhancement of Antibody Microarrays). ENSAM is based on two nanomaterials. The first is polystyrene nanoparticles incorporated with europium chelate (beta-diketone) and coated with streptavidin. The multiple fluorophores incorporated into each nanoparticle should increase signal obtained from a single binding event. The second nanomaterial is array surfaces of nanoporous silicon, which creates high capacity for antibody adsorption. Two antibody microarray assays were compared: ENSAM and use of streptavidin labeled with a nine-dentate europium chelate. Analyzing biotinylated prostate-specific antigen (PSA) spiked into human female serum, ENSAM yielded a 10-fold signal enhancement compared to the streptavidin-europium chelate. Similarly, we observed around 1 order of magnitude greater sensitivity for the ENSAM assay (limit of detection < or = 0.14 ng/mL, dynamic range > 10(5)) compared to the streptavidin-europium chelate assay (limit of detection < or = 0.7 ng/mL, dynamic range > 10(4)). Analysis of a titration series showed strong linearity of ENSAM ( R2 = 0.99 by linear regression). This work demonstrates the novel utility of nanoparticles with time-resolved fluorescence for signal enhancement of antibody microarrays, requiring as low as 100-200 zmol biotinylated PSA per microarray spot. In addition, proof of principle was shown for analyzing PSA in plasma obtained from patients undergoing clinical PSA-testing.
为提高抗体微阵列检测的灵敏度，我们开发了ENSAM（用于抗体微阵列信号增强的铕纳米颗粒）。ENSAM基于两种纳米材料。第一种是掺入铕螯合物（β-二酮）并包被链霉亲和素的聚苯乙烯纳米颗粒。每个纳米颗粒中掺入的多个荧光团应能增加单次结合事件获得的信号。第二种纳米材料是纳米多孔硅的阵列表面，其具有高抗体吸附能力。比较了两种抗体微阵列检测方法：ENSAM和使用九齿铕螯合物标记的链霉亲和素。在分析掺入人女性血清中的生物素化前列腺特异性抗原（PSA）时，与链霉亲和素-铕螯合物相比，ENSAM产生了10倍的信号增强。同样，我们观察到与链霉亲和素-铕螯合物检测方法（检测限≤0.7 ng/mL，动态范围>10⁴）相比，ENSAM检测方法的灵敏度提高了约1个数量级（检测限≤0.14 ng/mL，动态范围>10⁵）。滴定系列分析显示ENSAM具有很强的线性（线性回归R² = 0.99）。这项工作证明了具有时间分辨荧光的纳米颗粒在增强抗体微阵列信号方面的新用途，每个微阵列点只需低至100 - 200 zmol的生物素化PSA。此外，还展示了在接受临床PSA检测患者的血浆中分析PSA的原理证明。
