Section for Pharmaceutical Chemistry, Department of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway.
WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, Scotland, UK.
J Chromatogr A. 2021 Oct 11;1655:462490. doi: 10.1016/j.chroma.2021.462490. Epub 2021 Aug 31.
In the present work, a pair of molecularly imprinted polymers (MIPs) targeting distinct peptide targets were packed into trap columns and combined for automated duplex analysis of two low abundant small cell lung cancer biomarkers (neuron-specific enolase [NSE] and progastrin-releasing peptide [ProGRP]). Optimization of the on-line molecularly imprinted solid-phase extraction (MISPE) protocol ensured that the MIPs had the necessary affinity and selectivity towards their respective signature peptide targets - NLLGLIEAK (ProGRP) and ELPLYR (NSE) - in serum. Two duplex formats were evaluated: a physical mixture of the two MIPs (1:1 w/w ratio) inside a single trap column, and two separate MIP trap columns connected in series. Both duplex formats enabled the extraction of the peptides from serum. However, the trap columns in series gave superior extraction efficiency (85.8±3.8% and 49.1±6.7% for NLLGLIEAK and ELPLYR, respectively). The optimized protocol showed satisfactory intraday (RSD≤23.4 %) and interday (RSD≤14.6%) precision. Duplex analysis of NSE and ProGRP spiked into digested human serum was linear (R≥0.98) over the disease range (0.3-30 nM). The estimated limit of detection (LOD) and limit of quantification (LOQ) were 0.11 nM and 0.37 nM, respectively, for NSE, and 0.06 nM and 0.2 nM, respectively, for ProGRP. Both biomarkers were determined at clinically relevant levels. To the best of our knowledge, the present work is the first report of an automated MIP duplex biomarker analysis. It represents a proof of concept for clinically viable duplex analysis of low abundant biomarkers present in human serum or other biofluids.
在本工作中，针对两个低丰度小细胞肺癌生物标志物（神经元特异性烯醇化酶[NSE]和胃泌素释放肽前体[ProGRP]），将一对针对不同肽靶标的分子印迹聚合物（MIPs）包装到捕获柱中并组合用于自动化双分析。在线分子印迹固相萃取（MISPE）方案的优化确保了 MIP 对其各自特征肽靶标 - NLLGLIEAK（ProGRP）和 ELPLYR（NSE） - 在血清中有必要的亲和力和选择性。评估了两种双分析格式：一种是在单个捕获柱中混合两种 MIP（1:1w/w 比），另一种是串联连接的两个单独的 MIP 捕获柱。两种双分析格式均能够从血清中提取肽。然而，串联的捕获柱具有更高的提取效率（分别为 NLLGLIEAK 和 ELPLYR 的 85.8±3.8%和 49.1±6.7%）。优化的方案显示出令人满意的日内（RSD≤23.4%）和日间（RSD≤14.6%）精密度。NSE 和 ProGRP 双分析物加入消化后的人血清中呈线性（R≥0.98），疾病范围为 0.3-30 nM。NSE 的估计检测限（LOD）和定量限（LOQ）分别为 0.11 nM 和 0.37 nM，ProGRP 分别为 0.06 nM 和 0.2 nM。两种生物标志物均在临床相关水平上进行了测定。据我们所知，这是首次报道自动化 MIP 双生物标志物分析。它代表了在人血清或其他生物流体中低丰度生物标志物的临床可行双分析的概念验证。
