Yan Sijia, Ahmad Khan Zara, Li Sijie, Warden Antony R, Su Jing, Zhang Yu, Yu Youyi, Zhi Xiao, Ding Xianting
State Key Laboratory of Oncogenes and Related Genes, Institute for Personalized Medicine, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200030, China.
State Key Laboratory of Oncogenes and Related Genes, Institute for Personalized Medicine, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200030, China.
Biosens Bioelectron. 2021 Jul 1;183:113211. doi: 10.1016/j.bios.2021.113211. Epub 2021 Apr 6.
Advances in trace protein detection contribute to the early diagnosis of diseases and exploration of stem cell development. The pre-coated interface proximity extension reaction (PIPER) assay enables target protein detection at trace levels and was developed based on protein biomarker recognition using sets of three specific antibodies and the extension of antibody-bound nucleic acid chains in proximity, accompanied by amplification and reading of protein signals via real-time quantitative polymerase chain reaction (qPCR). Noise generated in binding reactions and enzymatic steps was decreased by transferring the liquid-liquid reactions onto a liquid-solid interface in glutaraldehyde-treated tubes pre-coated with antibodies. Nucleic acid sequences of oligo-antibody-based probes were designed for extension and qPCR without pre-amplification when binding to a target molecule. As a proof of concept, the PIPER assay was used to profile slight variations in crucial biomarkers, high-sensitivity C-reactive protein, and cardiac troponin I. The detection sensitivity of the assay for the biomarkers was 0.05 pg/mL (1.25 fM) in 10% human serum. In phosphate-buffered saline, the PIPER assay detected fewer than 10 protein molecules per μL. The simple, widely applicable PIPER assay can detect trace protein biomarkers with single-digit accuracy, making it appropriate for the development of clinical hypersensitive protein detection and single-cell protein detection technology.
痕量蛋白质检测技术的进步有助于疾病的早期诊断和干细胞发育的探索。预包被界面邻近延伸反应（PIPER）分析方法能够检测痕量水平的目标蛋白质，它基于使用三组特异性抗体识别蛋白质生物标志物以及邻近抗体结合核酸链的延伸而开发，同时通过实时定量聚合酶链反应（qPCR）对蛋白质信号进行扩增和读取。通过将液 - 液反应转移到预先用抗体包被的经戊二醛处理的管中的液 - 固界面上，减少了结合反应和酶促步骤中产生的噪音。基于寡核苷酸 - 抗体的探针的核酸序列在与目标分子结合时无需预扩增即可用于延伸和qPCR。作为概念验证，PIPER分析方法用于分析关键生物标志物、高敏C反应蛋白和心肌肌钙蛋白I的细微变化。该分析方法对生物标志物的检测灵敏度在10%人血清中为0.05 pg/mL（1.25 fM）。在磷酸盐缓冲盐水中，PIPER分析方法每微升检测到的蛋白质分子少于10个。这种简单且广泛适用的PIPER分析方法能够以个位数的精度检测痕量蛋白质生物标志物，使其适用于临床超敏蛋白质检测和单细胞蛋白质检测技术的开发。
