Yoo Minyoung, Yeo Woon-Seok
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University.
Anal Sci. 2016;32(10):1117-1121. doi: 10.2116/analsci.32.1117.
Prostate-specific antigen (PSA) is a well-known biomarker for prostate-cancer diagnosis. However, the serum PSA measurement alone is insufficient for accurate diagnoses because the correlation with cancer is weak within the gray zone-the biomarker level range wherein a clear-cut diagnosis is impossible. As such, accurate prostate cancer diagnosis has been supplemented by measurements of the ratio of two types of PSA: free PSA (fPSA) and complexed PSA (cPSA; α-1-antichymotrypsin-bound PSA). Herein, we describe a new method for measuring the ratio of these two types of PSA by using gold nanoparticles (AuNPs) and biochips. Both types of PSA in a sample are captured by the antibody immobilized on a biochip based on self-assembled monolayers on gold. fPSA and cPSA on the biochip are then distinguished by AuNPs that present antibodies against fPSA and cPSA, respectively. The presence of PSAs in a sample is detected with laser desorption/ionization time-of-flight mass spectrometry by observing reporter molecules, called amplification tags (Am-tags), on the AuNPs. One of the reporter molecules is an Am-tag without isotope labeling, and the other is a deuterium-labeled Am-tag (dAm-tag). These tags amplify mass signals so as to enhance the sensitivity of the method. A comparison of the mass intensities between the Am-tag and dAm-tag signals allows the determination of the ratio between fPSA and cPSA. We validated the selective measurement of fPSA and cPSA at different ratios in 50, 75, and 100 pM of total PSA (fPSA + cPSA) solutions corresponding to the gray zone in prostate-cancer diagnosis (4 - 10 ng/mL). Finally, the two types of PSA were spiked in fetal bovine serum at various ratios, and our strategy greatly afforded their accurate ratios as spiked based on a constructed calibration curve. These results clearly indicate that the strategy is applicable to human serum as a diagnostic and prognostic assay for prostate cancer.
前列腺特异性抗原（PSA）是一种广为人知的用于前列腺癌诊断的生物标志物。然而，仅血清PSA测量不足以进行准确诊断，因为在灰色区域（即无法进行明确诊断的生物标志物水平范围）内，其与癌症的相关性较弱。因此，通过测量两种类型的PSA：游离PSA（fPSA）和复合PSA（cPSA；α-1-抗糜蛋白酶结合的PSA）的比值来补充前列腺癌的准确诊断。在此，我们描述了一种使用金纳米颗粒（AuNPs）和生物芯片测量这两种类型PSA比值的新方法。样品中的两种类型的PSA均被固定在基于金上自组装单层的生物芯片上的抗体捕获。然后，通过分别呈现抗fPSA和cPSA抗体的AuNPs来区分生物芯片上的fPSA和cPSA。通过观察AuNPs上称为扩增标签（Am-标签）的报告分子，利用激光解吸/电离飞行时间质谱法检测样品中PSA的存在。其中一种报告分子是未进行同位素标记的Am-标签，另一种是氘标记的Am-标签（dAm-标签）。这些标签放大质量信号以提高该方法的灵敏度。比较Am-标签和dAm-标签信号之间的质量强度可确定fPSA和cPSA之间的比值。我们在前列腺癌诊断灰色区域（4 - 10 ng/mL）对应的50、75和100 pM总PSA（fPSA + cPSA）溶液中，验证了以不同比值对fPSA和cPSA的选择性测量。最后，将两种类型的PSA以不同比例添加到胎牛血清中，并且我们的策略基于构建的校准曲线极大地提供了添加后的准确比例。这些结果清楚地表明，该策略适用于作为前列腺癌诊断和预后分析的人血清。
