Generation of highly specific aptamers via micromagnetic selection.
Generation of highly specific aptamers via micromagnetic selection.
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DOI：
10.1021/ac900759k
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发表时间：
2009-07-01
影响因子：
7.4
通讯作者：
Soh, H. Tom
中科院分区：
文献类型：
--
作者：
Qian, Jiangrong;Lou, Xinhui;Zhang, Yanting;Xiao, Yi;Soh, H. Tom
适体是以高亲和力和特异性结合靶分子的基于核酸的试剂。然而，从随机组合文库（例如，SELEX）通常是劳动密集型和耗时的。最近的研究表明，微流体SELEX（M-SELEX）技术可以通过使用非常少量的靶分子实现高度严格的选择条件来加速适体分离。我们在这里提出了一种替代的M-SELEX方法，该方法采用一次性微流控芯片快速生成具有高亲和力和特异性的适体。微磁分离（MMS）芯片集成了微制造的铁磁结构，可在其微通道内重复产生大的磁场梯度，有效地捕获磁珠结合的适体。MMS装置的操作是容易的、稳健的，并且证明珠的高回收率（99.5%），使得可以使用皮摩尔量的靶分子。重要的是，该装置在去除弱结合和未结合的ssDNA以快速富集靶特异性适体方面表现出卓越的分离效率。作为一个模型，我们在这里展示了在三轮阳性选择中产生针对链霉亲和素的DNA适体。我们通过在同一装置中对牛血清白蛋白（BSA）进行一轮阴性选择，进一步增强了所选适体的特异性。所得适体显示出对链霉亲和素的解离常数范围为25至65 nM，但对BSA的亲和力可忽略不计。由于广泛的分子靶点可以很容易地结合在磁珠上，基于MMS的SELEX应该为快速生成特异性适体提供通用平台。
Aptamers are nucleic acid-based reagents that bind to target molecules with high affinity and specificity. However, methods for generating aptamers from random combinatorial libraries (e.g., SELEX) are often labor-intensive and time-consuming. Recent studies suggest that microfluidic SELEX (M-SELEX) technology can accelerate aptamer isolation by enabling highly stringent selection conditions through the use of very small amounts of target molecules. We present here an alternative M-SELEX method, which employs a disposable microfluidic chip to rapidly generate aptamers with high affinity and specificity. The Micro-Magnetic Separation (MMS) chip integrates microfabricated ferromagnetic structures to reproducibly generate large magnetic field gradients within its microchannel that efficiently trap magnetic bead-bound aptamers. Operation of the MMS device is facile, robust and demonstrates high recovery of the beads (99.5%), such that picomolar amounts of target molecule can be used. Importantly, the device demonstrates exceptional separation efficiency in removing weakly-bound and unbound ssDNA to rapidly enrich target-specific aptamers. As a model, we demonstrate here the generation of DNA aptamers against streptavidin in three rounds of positive selection. We further enhanced the specificity of the selected aptamers via a round of negative selection in the same device against bovine serum albumin (BSA). The resulting aptamers displayed dissociation constants ranging from 25 to 65 nM for streptavidin but negligible affinity for BSA. Since a wide spectrum of molecular targets can be readily conjugated on magnetic beads, MMS-based SELEX should provide a general platform for rapid generation of specific aptamers.
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影响因子：
3.9
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