Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269, United States.
Department of Surgery and Neag Cancer Center, UConn Health, Farmington, Connecticut 06232, United States.
Anal Chem. 2020 Jun 16;92(12):8021-8025. doi: 10.1021/acs.analchem.0c01507. Epub 2020 May 27.
We report here a low-cost electrochemical immunoarray with unprecedented sensitivity in the sub-zeptomole range with up to 5 log-decades dynamic range for accurate, multiplexed protein determinations. The microfluidic array features eight carbon sensors coated with a dense layer of 5 nm gold-nanoparticles derivatized with primary antibodies. Analyte proteins are captured by secondary antibody-poly-HPR (horseradish peroxidase) bioconjugates containing 400 HRP enzyme labels, with amplified amperometric peaks developed using HO activator and hydroquinone mediator. Prostate cancer biomarkers prostate specific antigen (PSA), vascular endothelial growth factor-D (VEGF-D), ETS-related gene protein (ERG), and insulin-like growth factor-1 (IGF-1) were measured simultaneously with sub-fg/mL LODs (0.08-0.22 zmol). These proteins were determined in serum of postprostatectomy cancer patients which had much lower levels than prostate cancer patients without surgery. This immunoassay protocol makes thousands of low-abundance proteins accessible to quantitative measurements down to zeptomole levels.
我们在此报告一种具有成本效益的电化学免疫微阵列，其灵敏度达到亚zeptomole 级，动态范围高达 5 个对数 decades，可实现准确、多重蛋白质测定。该微流控阵列具有 8 个碳传感器，其表面涂有一层密集的 5nm 金纳米粒子，金纳米粒子通过与一级抗体衍生化。分析物蛋白通过含有 400 个辣根过氧化物酶（HRP）酶标签的二级抗体-多-HPR（辣根过氧化物酶）生物缀合物被捕获，使用 HO 激活剂和对苯二酚介体开发放大的安培峰值。同时测量前列腺癌生物标志物前列腺特异性抗原（PSA）、血管内皮生长因子-D（VEGF-D）、ETS 相关基因蛋白（ERG）和胰岛素样生长因子-1（IGF-1），其检测限（LOD）低至亚 fg/mL（0.08-0.22 zmol）。这些蛋白质在前列腺癌手术后患者的血清中进行了测定，其水平远低于未接受手术的前列腺癌患者。这种免疫分析方案使得数千种低丰度蛋白质能够进行定量测量，达到zeptomole 水平。
