Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga , 3359 Mississauga Road, Mississauga, ON, L5L 1C6, Canada.
Anal Chem. 2014 Oct 21;86(20):10331-9. doi: 10.1021/ac502677n. Epub 2014 Sep 29.
Paper-based diagnostic assays are gaining increasing popularity for their potential application in resource-limited settings and for point-of-care screening. Achievement of high sensitivity with precision and accuracy can be challenging when using paper substrates. Herein, we implement the red-green-blue color palette of a digital camera for quantitative ratiometric transduction of nucleic acid hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). A nonenzymatic and reagentless means of signal enhancement for QD-FRET assays on paper substrates is based on the use of dry paper substrates for data acquisition. This approach offered at least a 10-fold higher assay sensitivity and at least a 10-fold lower limit of detection (LOD) as compared to hydrated paper substrates. The surface of paper was modified with imidazole groups to assemble a transduction interface that consisted of immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs was responsible for a FRET-sensitized emission from the acceptor dye, which served as an analytical signal. A hand-held UV lamp was used as an excitation source and ratiometric analysis using an iPad camera was possible by a relative intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an important framework for the integration of QD-FRET methods with digital imaging for a ratiometric transduction of nucleic acid hybridization on a paper-based platform.
基于纸的诊断检测因其在资源有限环境中的潜在应用和在现场进行筛选的能力而日益受到关注。在使用纸质基质时，实现高精度和准确性的高灵敏度可能具有挑战性。在此，我们利用数码相机的红绿蓝调色板，使用固定化量子点（QD）作为荧光共振能量转移（FRET）中的供体，在基于纸的平台上实现核酸杂交的定量比比率转换。一种非酶和无试剂的信号增强方法是基于在纸基底物上进行 QD-FRET 测定时使用干燥的纸基底物进行数据采集。与水合纸基相比，这种方法提供了至少 10 倍更高的测定灵敏度和至少 10 倍更低的检测限（LOD）。纸的表面用咪唑基团进行修饰，组装了一个转导界面，该界面由固定化 QD-探针寡核苷酸缀合物组成。发绿光的 QD（gQD）作为供体，Cy3 作为受体。使 Cy3 受体染料靠近固定化 gQD 表面的杂交事件导致来自受体染料的 FRET 敏化发射，该发射充当分析信号。手持紫外灯用作激发源，并且通过相对强度分析数字相机的红色（Cy3 光致发光（PL））和绿色（gQD PL）颜色通道，可实现使用 iPad 相机的比分析。对于使用 iPad 相机进行数字成像，夹心格式测定的 LOD 为 450 fmol，动态范围跨越 2 个数量级，而荧光显微镜检测平台的 LOD 为 30 fmol，动态范围跨越 3 个数量级。通过检测单核苷酸多态性的对比率为 60:1，证明了杂交测定的选择性。这项工作为将 QD-FRET 方法与数字成像相结合提供了重要框架，用于在基于纸的平台上进行核酸杂交的比比率转换。
