Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
Bioconjug Chem. 2010 Oct 20;21(10):1820-6. doi: 10.1021/bc100169y.
We report a simple fluidic system that can purify and concentrate diagnostic biomarkers through the capture and triggered release of stimuli-responsive polymer-antibody conjugates at porous membranes that are grafted with the same stimuli-responsive polymer. This technique is applied here to the capture and detection of a model streptavidin antigen and subsequently to clinical ranges of the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) from spiked human plasma. The carboxyl end-groups of semi-telechelic poly(N-isopropylacrylamide) (pNIPAAm) synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization were modified with tetrafluorophenol to yield amine-reactive ester groups for conjugation to amine groups of anti-streptavidin and anti-PfHRP2 antibodies. Stimuli-responsive membranes were constructed from 1.2 μm pore-size, hydroxylated, nylon-6,6 filters (Loprodyne, from Pall Corporation). The surface hydroxyl groups on the filters were conjugated to a 2-ethylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (EMP) RAFT chain transfer agent, and the surface-grafted pNIPAAm was obtained by subsequent polymerization. The number average molecular weight (Mn) and polydispersity indices (PDI) of the surface grafts were characterized, and membranes with either 4100 and 8400 dalton pNIPAAm grafts showed greater than 80% anti-streptavidin capture efficiency. The 8400 dalton-graft membrane showed the highest release efficiency, and it was demonstrated that at 0.2 nM starting concentration the streptavidin could be concentrated approximately 40-fold by releasing into a small 50 μL volume. This concentrator system was applied to the capture and concentration of the PfHRP2 antigen, and results showed that the PfHRP2 antigen could be processed and detected at clinically relevant concentrations of this malaria biomarker.
我们报告了一种简单的流体系统，该系统可以通过在接枝相同响应性聚合物的多孔膜上捕获和触发刺激响应性聚合物-抗体缀合物的释放来纯化和浓缩诊断生物标志物。 该技术在这里应用于捕获和检测模型链霉亲和素抗原，随后应用于临床相关浓度的疟疾抗原恶性疟原虫环子氨酸-富含蛋白 2（PfHRP2）从掺杂的人血浆中。 通过可逆加成-断裂链转移（RAFT）聚合合成的半遥爪聚（N-异丙基丙烯酰胺）（pNIPAAm）的羧基端基被四氟苯酚修饰，得到用于与抗链霉亲和素和抗 PfHRP2 抗体的胺基反应的酯基。 刺激响应性膜由 1.2μm 孔径、羟基化的尼龙-6,6 过滤器（Loprodyne，来自 Pall Corporation）构建。 过滤器表面上的羟基与 2-乙硫基硫代羰基硫代-2-甲基丙酸（EMP）RAFT 链转移剂连接，随后通过聚合得到表面接枝的 pNIPAAm。 表面接枝的数均分子量（Mn）和多分散指数（PDI）进行了表征，并且具有 4100 和 8400 道尔顿 pNIPAAm 接枝的膜显示出大于 80％的抗链霉亲和素捕获效率。 8400 道尔顿接枝膜显示出最高的释放效率，并且证明在 0.2 nM 起始浓度下，通过释放到 50μL 小体积中，链霉亲和素可以浓缩约 40 倍。 该浓缩器系统应用于 PfHRP2 抗原的捕获和浓缩，结果表明可以处理和检测到该疟疾生物标志物的临床相关浓度的 PfHRP2 抗原。
