A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.
A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.
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用于快速分离蛋白质生物标志物的刺激响应二元试剂系统。
DOI：
10.1021/acs.analchem.6b01961
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发表时间：
2016
影响因子：
7.4
通讯作者：
Lai,JamesJ
中科院分区：
文献类型：
--
作者：
Nehilla,BarrettJ;Hill,JohnJ;Srinivasan,Selvi;Chen,Yen-Chi;Schulte,ThomasH;Stayton,PatrickS;Lai,JamesJ
磁微珠具有快速分离的特点，被广泛应用于生物分子和细胞分离研究实验室，临床诊断分析和细胞治疗制造。然而，微米级的颗粒直径会影响生物标志物的识别，从而导致较长的孵育时间和显著的试剂需求。本文提出了一种刺激响应型二元试剂系统，该系统结合了纳米尺度的高效生物标志物识别和微尺度的快速磁分离。该系统由磁性纳米粒子和聚合物-抗体（Ab）偶联物组成，这些偶联物在温度刺激下从亲水性纳米级试剂转变为微尺度聚集体。在生物标志物分离动力学、测定速度和试剂需求方面，二元试剂系统与ab标记的dynabead进行了基准比较。表面等离子体共振（SPR）测量表明，聚合物偶联没有显著改变Ab的结合亲和力或动力学。ELISA分析表明，未偶联的Ab、聚合物- Ab偶联物和Ab标记的dynabead具有相似的平衡解离常数（Kd），约2 nM。然而，二元试剂系统从加标血清标本（150 pg/mL）中分离HIV p24抗原的速度比Dynabeads快得多，其结合时间缩短了数十分钟，或缩短了约30-50%的总测定时间。二元试剂体系表现出更好的性能，因为Ab分子没有共轭到大的固体微粒表面。这种刺激反应二元试剂系统说明了纳米级试剂在分子和细胞分离研究和临床应用中的潜在优势。
Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays, and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microscale benefits of rapid magnetic separation. This system comprises magnetic nanoparticles and polymer–antibody (Ab) conjugates that transition from hydrophilic nanoscale reagents to microscale aggregates in response to temperature stimuli. The binary reagent system was benchmarked against Ab-labeled Dynabeads in terms of biomarker isolation kinetics, assay speed, and reagent needs. Surface plasmon resonance (SPR) measurements showed that polymer conjugation did not significantly alter the Ab’s binding affinity or kinetics. ELISA analysis showed that the unconjugated Ab, polymer–Ab conjugates, and Ab-labeled Dynabeads exhibited similar equilibrium dissociation constants (Kd), ∼2 nM. However, the binary reagent system isolated HIV p24 antigen from spiked serum specimens (150 pg/mL) much more quickly than Dynabeads, which resulted in shorter binding times by tens of minutes, or about 30–50% shorter overall assay times. The binary reagent system showed improved performance because the Ab molecules were not conjugated to large, solid microparticle surfaces. This stimuli-responsive binary reagent system illustrates the potential advantages of nanoscale reagents in molecule and cell isolations for both research and clinical applications.