Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy
Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy
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DOI：
10.1073/pnas.1904770116
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发表时间：
2019-09-24
影响因子：
11.1
通讯作者：
Cunningham, Brian T.
中科院分区：
文献类型：
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作者：
Canady, Taylor D.;Li, Nantao;Cunningham, Brian T.
循环外泌体 microRNA (miR) 代表了一类新的基于血液的癌症液体活检生物标志物。在非常低的浓度下检测 miR 并进行单碱基区分，而不需要复杂的设备、大体积或复杂的样品处理是一个挑战。为了解决这个问题，我们提出了一种对目标 miR 序列具有高度特异性的方法，并且能够以高信噪比提供单个目标分子的“数字”分辨率。金纳米颗粒标签是用热力学优化的核酸立足点探针制备的，当与目标 miR 序列结合时，会取代探针保护寡核苷酸并显示捕获序列，该捕获序列用于选择性地将目标-探针-纳米颗粒复合物下拉至光子晶体 (PC) 生物传感器表面。通过将纳米颗粒标签的表面等离激元共振波长与 PC 纳米结构的共振波长相匹配，来自 PC 的反射光强度会因每个纳米颗粒的存在而被局部显着淬灭，从而实现一种我们称为光子谐振器吸收显微镜 (PRAM) 的生物传感器显微镜。纳米颗粒标签捕获的动态 PRAM 成像可在 2 小时动态辨别测定中实现直接 100-aM 的检测限和单碱基错配选择性。 PRAM 测定表明超灵敏（
Circulating exosomal microRNA (miR) represents a new class of blood-based biomarkers for cancer liquid biopsy. The detection of miR at a very low concentration and with single-base discrimination without the need for sophisticated equipment, large volumes, or elaborate sample processing is a challenge. To address this, we present an approach that is highly specific for a target miR sequence and has the ability to provide "digital" resolution of individual target molecules with high signal-to-noise ratio. Gold nanoparticle tags are prepared with thermodynamically optimized nucleic acid toehold probes that, when binding to a target miR sequence, displace a probe-protecting oligonucleotide and reveal a capture sequence that is used to selectively pull down the target-probe-nanoparticle complex to a photonic crystal (PC) biosensor surface. By matching the surface plasmon-resonant wavelength of the nanoparticle tag to the resonant wavelength of the PC nanostructure, the reflected light intensity from the PC is dramatically and locally quenched by the presence of each individual nanoparticle, enabling a form of biosensor microscopy that we call Photonic Resonator Absorption Microscopy (PRAM). Dynamic PRAM imaging of nanoparticle tag capture enables direct 100-aM limit of detection and single-base mismatch selectivity in a 2-h kinetic discrimination assay. The PRAM assay demonstrates that ultrasensitivity (