Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
ACS Nano. 2012 Aug 28;6(8):6776-85. doi: 10.1021/nn3015008. Epub 2012 Jul 24.
There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide was prepared by reversible addition-fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity.
需要开发简单而稳健的生物标志物和抗原纯化和富集策略，使其与当前快速诊断模式兼容。在这里，提出了一种响应性纳米粒子系统，用于从加标混合血浆中对模型抗原进行多重磁富集和无仪器横向流动条带检测。集成试剂系统允许纯化和富集金标记的生物标志物半夹心，可以直接应用于横向流动测试条。通过可逆加成-断裂链转移聚合制备了具有热响应性聚（N-异丙基丙烯酰胺）（pNIPAm）段和由 NIPAm-co-N，N-二甲基氨基乙基丙烯酰胺组成的金结合嵌段的线性两亲嵌段共聚物。两亲嵌段共聚物用于功能化金纳米粒子（AuNPs），随后进行生物缀合，得到具有热响应性的 pNIPAm-AuNPs，其共修饰有链霉亲和素。这些 AuNPs 有效地与生物素化捕获抗体试剂复合，该试剂结合到加标混合血浆样品中纳摩尔级的泛醛缩酶和恶性疟原虫富组氨酸蛋白 2（PfHRP2）。然后使用 10nm 热响应磁性纳米粒子纯化和富集金标记的生物标志物半夹心，该纳米粒子也用 pNIPAm 修饰。当施加热刺激并结合磁场时，AuNP 半夹心与涂有 pNIPAm 的氧化铁纳米粒子的共聚集会产生大的聚集体，这些聚集体可以有效地磁泳并从大量血清中分离出来。从加标混合血浆样品中纯化的生物标志物可以浓缩 50 倍到小体积，并直接应用于商业的多重横向流动条带，从而显著提高信噪比和测试灵敏度。
