Simple fluidic system for purifying and concentrating diagnostic biomarkers using stimuli-responsive antibody conjugates and membranes.
Simple fluidic system for purifying and concentrating diagnostic biomarkers using stimuli-responsive antibody conjugates and membranes.
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DOI：
10.1021/bc100169y
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发表时间：
2010-10-20
影响因子：
4.7
通讯作者：
Stayton, Patrick S.
中科院分区：
文献类型：
--
作者：
Golden, Allison L.;Battrell, Charles F.;Pennell, Sean;Hoffman, Allan S.;Lai, James J.;Stayton, Patrick S.
我们报告了一个简单的流体系统，可以纯化和浓缩诊断生物标志物，通过捕获和触发释放的刺激响应性聚合物-抗体缀合物在多孔膜接枝有相同的刺激响应性聚合物。该技术应用于捕获和检测模型链霉亲和素抗原，随后应用于从加标人血浆中捕获和检测疟疾抗原恶性疟原虫富组氨酸蛋白2（PfHRP 2）的临床范围。通过可逆加成断裂链转移（RAFT）聚合合成的聚（N-异丙基丙烯酰胺）（pNIPAAm）的半遥爪末端羧基用四氟苯酚修饰以产生胺反应性酯基团用于与抗链霉亲和素和抗PfHRP 2抗体的胺基缀合。刺激响应膜由1.2 μm孔径的羟基化尼龙6，6过滤器（Loprodyne，来自Pall Corporation）构建。将滤膜表面羟基与2-乙基硫烷基硫代羰基硫烷基-2-甲基丙酸（EMP）RAFT链转移剂偶联，通过后续聚合得到表面接枝的pNIPAAm。表征表面接枝物的数均分子量（Mn）和多分散指数（PDI），并且具有4100和8400 Mn pNIPAAm接枝物的膜显示大于80%的抗链霉亲和素捕获效率。8400分子量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，通过释放到小的50 μl体积中，链霉亲和素可以浓缩约40倍。将该浓缩器系统应用于恶性疟原虫HRP 2抗原的捕获和浓缩，结果显示PfHRP 2抗原可以在该疟疾生物标志物的临床相关浓度下被处理和检测。
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 semi-telechelic end carboxyl groups of 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 Mn pNIPAAm grafts showed greater than 80% anti-streptavidin capture efficiency. The 8400 molecular weight-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 Plasmodium falciparum HRP2 antigen and results showed that the PfHRP2 antigen could be processed and detected at clinically-relevant concentrations of this malaria biomarker.
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影响因子：
3.9
作者：
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通讯作者：
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DOI：
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发表时间：
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