Mazzarotta Alessia, Caputo Tania Mariastella, Battista Edmondo, Netti Paolo Antonio, Causa Filippo
Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, L.go Barsanti e Matteucci, 53, 80125 Naples, Italy.
Interdisciplinary Research Centre on Biomaterials (CRIB), Università degli Studi di Napoli "Federico II", P.le Tecchio 80, 80125 Naples, Italy.
Sensors (Basel). 2021 Nov 18;21(22):7671. doi: 10.3390/s21227671.
Herein we describe the development of a mix-read bioassay based on a three-dimensional (3D) poly ethylene glycol-(PEG)-hydrogel microparticles for the detection of oligonucleotides in complex media. The key steps of hydrogels synthesis and molecular recognition in a 3D polymer network are elucidated. The design of the DNA probes and their density in polymer network were opportunely optimized. Furthermore, the diffusion into the polymer was tuned adjusting the polymer concentration and consequently the characteristic mesh size. Upon parameters optimization, 3D-PEG-hydrogels were synthetized in a microfluidic system and provided with fluorescent probe. Target detection occurred by double strand displacement assay associated to fluorescence depletion within the hydrogel microparticle. Proposed 3D-PEG-hydrogel microparticles were designed for miR-143-3p detection. Results showed 3D-hydrogel microparticles with working range comprise between 10-10 M, had limit of detection of 30 pM and good specificity. Moreover, due to the anti-fouling properties of PEG-hydrogel, the target detection occurred in human serum with performance comparable to that in buffer. Due to the approach versatility, such design could be easily adapted to other short oligonucleotides detection.
在此，我们描述了一种基于三维（3D）聚乙二醇（PEG）水凝胶微粒的混合读取生物测定法的开发，用于检测复杂介质中的寡核苷酸。阐明了水凝胶合成和三维聚合物网络中分子识别的关键步骤。对DNA探针的设计及其在聚合物网络中的密度进行了适当优化。此外，通过调节聚合物浓度进而调整特征网孔尺寸来调控向聚合物中的扩散。在参数优化后，在微流控系统中合成了3D-PEG水凝胶，并配备了荧光探针。通过与水凝胶微粒内荧光消耗相关的双链置换测定法进行目标检测。所提出的3D-PEG水凝胶微粒设计用于检测miR-143-3p。结果表明，3D水凝胶微粒的工作范围在10⁻¹⁰ M之间，检测限为30 pM，且具有良好的特异性。此外，由于PEG水凝胶的抗污染特性，在人血清中进行目标检测时的性能与在缓冲液中相当。由于该方法具有通用性，这种设计可以很容易地适用于其他短寡核苷酸的检测。
