Ultrasensitive Fluorescent miRNA Biosensor Based on a “Sandwich” Oligonucleotide Hybridization and Fluorescence Resonance Energy Transfer Process Using an Ln(III)-MOF and Ag Nanoparticles for Early Cancer Diagnosis: Application of Central Composite Design

Abstract

Herein, a novel, rapid, highly sensitive, and selective fluorescent biosensor is presented, which is designed based on “sandwich-type” hybridization of oligonucleotides and the fluorescence resonance energy transfer (FRET) strategy. It senses and determines the MicroRNA-155 (miRNA-155) expression levels as a cancer biomarker. In this study, a modified La­(III)-metal–organic framework­(MOF) and silver nanoparticles (Ag NPs) were used as the energy donor–acceptor pairs in fluorescence quenching through the FRET process. La­(III)-MOF was synthesized and then modified by glutaraldehyde as a cross-linking agent. The Ag NPs were also prepared, and then, the surface of both was conjugated with different 5′-amino-labeled ssDNA strands (aptamers). These prepared nanoprobes were characterized by various physicochemical techniques such as X-ray diffraction, energy-dispersive X-ray spectrometry, Fourier transform infrared, field emission scanning electron microscopy, UV–vis spectroscopy, elemental mapping, and gel electrophoresis. To optimize the detection conditions, several factors affecting biosensor performance were assessed by one variable-at-a-time and central composite design methods. Under optimum conditions, this “turn-off” fluorescent biosensor could detect and determine as low as 0.04 ppb (ng. mL–1) or 5.5 fM of the miRNA-155 biomarker. Therefore, this biosensor provides highly promising potential for lung and breast cancer diagnosis.

Bioassay Applications of Aptamer-Functionalized Rare Earth Nanomaterials

“…由于 稀土离子通常通过能量/电子转移过程被周围的配体激 发, 稀土基 MOFs 中的有机配体在吸收激发光的能量后, 通过非辐射过程把能量转移到稀土离子, 从而可以产生 发光现象. 因此, 以稀土离子为金属节点的 MOFs 在光 学传感领域的应用也逐渐成为了研究的热点 [14,33] . 由于 结构和功能的可调性, 通过对稀土基 MOFs 中的稀土离 子和有机配体的调节设计的稀土基 MOFs 具有良好的发 光特性 [34] .…”

“…该特 点主要来源于以下两个原因: 其一, 由于稀土离子嵌入 MOFs 高度有序的有机配体框架中避免了由于聚集引起 的自猝灭, 从而使稀土基 MOFs 的发光增强 [36] . 其二, 稀土基 MOFs 的高比表面积能够实现核酸适配体的高负 载, 从而显著增加与靶标物结合的有效活性位点的数 量, 促进核酸适配体与靶标物结合的效率, 达到降低纳 米荧光探针的检测限的效果, 使纳米荧光探针的传感性 能得到提升 [14] . 总之, 基于稀土基 MOFs 设计的纳米荧 光探针在生物检测领域具有巨大潜力, 并在提升纳米荧 光探针性能方面有着独特的优势.…”

“…其次, 核酸适配体的生物相容性好、性质稳定, 对纳米 材料的功能化能够降低纳米材料的毒性同时还能增强 纳米材料的血液循环能力 [10] . 最重要的是, 核酸适配体 的结合目标广, 可以用于多种目标物的测定, 包括金属 离子 [11] 、 小分子(如三磷酸腺苷(ATP) [12] )、 生物大分子(如 蛋白质 [13] 、核酸 [14] )、细胞 [15] 以及细菌 [16] , 极大地拓展 了纳米荧光探针的检测范围. 因此, 基于核酸功能化的 稀土基纳米材料构建的纳米荧光探针不仅稳定性和生 物相容性好, 并且能够极大增强与靶标物的结合能力, 从而实现在复杂生物体系中对靶标物的检测.…”

Nanomaterial-Based Fluorescence Resonance Energy Transfer (FRET) and Metal-Enhanced Fluorescence (MEF) to Detect Nucleic Acid in Cancer Diagnosis

“…A higher binding force between the target miRNA-21 and the ssDNA probe was enhanced through the improved internalization process due to the hybridization of the endogenous miRNA and FA-functionalized MoS 2 nanosheets (FAM)-ssDNA, and a fast fluorescence signal was confirmed due to the separation of the dye-labeled ssDNA probe from the MoS 2 nanosheets. Afzalinia et al used an La (III) metal-organic framework (MOF) and silver NPs (AgNPs) as the energy donor-acceptor pair in fluorescence quenching to detect the atypically expressed miRNA-155 in patients with breast or lung cancer [69]. The use of MOFs with large surface area significantly improved the miRNA detection performance by increasing the number of aptamers attached to the probe surface.…”

Biosensor Technology in Diagnostics and Mechanistic Studies of Epilepsy

“…In this case the target analyte was miRNA connected to lumg cancer, although no actual samples were assessed. Lung cancer detection was also the recent focus of another fluorescencebased assay [25]. In order to detect miRNA-155 expression levels a modified La(III)-metal-organic framework(MOF) and silver nanoparticles (Ag NPs) were used as the energy donor-acceptor pairs in fluorescence quenching through the FRET process.…”