Department of Chemistry, Iowa State University, Ames, Iowa 50011, United States.
Anal Chem. 2022 Aug 30;94(34):11949-11956. doi: 10.1021/acs.analchem.2c02979. Epub 2022 Aug 16.
Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between single-nucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequence-specific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.
核酸检测广泛应用于从生物样本中扩增和定量核酸。聚合酶链反应（PCR）虽然广受欢迎，但需要昂贵的热循环仪来精确控制温度。环介导等温扩增（LAMP）可以在恒温下实现核酸的高灵敏度、快速和低成本扩增。LAMP 检测通常依赖于双链 DNA 结合染料或缺乏序列选择性的金属指示剂。分子信标（MBs）是发夹状寡核苷酸探针，其在 LAMP 中的序列特异性提供了区分单核苷酸多态性（SNP）的能力。数字液滴 LAMP（ddLAMP）能够进行大量独立的 LAMP 反应，并对靶 DNA 序列进行定量。然而，ddLAMP 的一个主要挑战是需要昂贵的液滴发生器来形成均匀的微液滴。在本研究中，我们首次证明了可以将 3D 打印的液滴生成平台与具有 MB 作为序列特异性探针的 LAMP 测定法相结合。设计了低成本的 3D 打印液滴发生器系统，并在形成单分散 ddLAMP assay-in-oil 微液滴中证明了其可定制性。此外，还展示了一种基于智能手机的成像系统，以增加在护理点应用中的可及性。MB-ddLAMP 测定法可在不同扩增温度下区分两个 SNP，为 SNP 序列特异性、敏感和准确 DNA 定量提供了有用的平台。这项工作扩展了 MB 在 ddLAMP 中的应用，用于 SNP 检测中的定量研究，并利用 3D 打印技术的可定制性来优化油包水微液滴的均匀性、大小和体积。
