Key Laboratory of Chem-Biosensing, Anhui Province, Wuhu 241000, PR China; Key Laboratory of Functional Molecular Solids, Anhui Province, Wuhu 241000, PR China; College of Chemistry and Materials Science, Center for Nano Science and Technology, Anhui Normal University, Wuhu 241000, PR China.
Key Laboratory of Chem-Biosensing, Anhui Province, Wuhu 241000, PR China; Key Laboratory of Functional Molecular Solids, Anhui Province, Wuhu 241000, PR China; College of Chemistry and Materials Science, Center for Nano Science and Technology, Anhui Normal University, Wuhu 241000, PR China; State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China.
Biosens Bioelectron. 2015 Dec 15;74:931-8. doi: 10.1016/j.bios.2015.07.052. Epub 2015 Jul 23.
Due to terbium's unique photophysical properties, nucleic-acid-sensitized terbium (DNA/Tb(3+)) bioluminescent system becomes a potential candidate for the fabrication of DNA biosensors. However, the low sensitivity of DNA/Tb(3+) bioluminescent system limits its development. In this paper, a strategy combining autocatalytic multi-cycle-amplification (including exonuclease III (exo III)-aided and Zn(2+)-requiring DNAzyme-assisted target recycling amplifications) and magnetic nanoparticles assisted-background-lowering to improve the sensitivity of DNA/Tb(3+) bioluminescent system is presented for sensitive detection of target DNA (tDNA). The DNA/Tb(3+) bioluminescent system was investigated by ultraviolet-visible (UV-vis) absorption and luminescence spectra. The possible conjugation mechanism and mode of DNA with Tb(3+) were discussed. The autocatalytic multi-cycle-amplification effect was investigated by the comparison of the luminescence. The carboxylation-functionalized Fe3O4-magnetic nanoparticles (MNPs) were characterized and its role in background lowering was proved. As a result, with the designed protocol, the detection limit for the tDNA detection reached a low level to aM, which is especially exciting for the DNA/Tb(3+) bioluminescent system. In the process, due to the separation effect of MNPs, the assay solution was purified to avoid the nonspecific luminescence of DNA/Tb(3+), not only lowering the background signal greatly (about five times lower than that without the use of MNPs but also improving the reproducibility and stability. We hope that our attempt in this field will not only extend the application of DNA/Tb(3+) luminescent system in biosensing areas but also open the road to adaptation of the protocols to other related analytes.
由于铽独特的光物理性质，核酸敏化铽（DNA/Tb(3+)）生物发光系统成为制备 DNA 生物传感器的潜在候选者。然而，DNA/Tb(3+) 生物发光系统的低灵敏度限制了其发展。本文提出了一种结合自动催化多循环扩增（包括外切酶 III（exo III）辅助和 Zn(2+) 依赖的 DNA 酶辅助靶标循环扩增）和磁性纳米粒子辅助背景降低的策略，以提高 DNA/Tb(3+) 生物发光系统的灵敏度，用于灵敏检测靶 DNA（tDNA）。通过紫外-可见（UV-vis）吸收和发光光谱研究了 DNA/Tb(3+) 生物发光系统。讨论了 DNA 与 Tb(3+) 的可能结合机制和方式。通过比较发光研究了自动催化多循环扩增效果。对羧基化功能化 Fe3O4-磁性纳米粒子（MNPs）进行了表征，并证明了其在降低背景方面的作用。结果，通过设计的方案，tDNA 的检测限达到了 aM 级，这对 DNA/Tb(3+) 生物发光系统来说尤其令人兴奋。在这个过程中，由于 MNPs 的分离效果，对检测溶液进行了纯化，以避免 DNA/Tb(3+) 的非特异性发光，不仅大大降低了背景信号（约比不使用 MNPs 低五倍），而且提高了重现性和稳定性。我们希望我们在这一领域的尝试不仅将扩展 DNA/Tb(3+) 发光系统在生物传感领域的应用，而且为适应其他相关分析物的方案开辟道路。
