Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200030, China.
Department of Infectious Diseases, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China.
Biosensors (Basel). 2024 Jul 29;14(8):370. doi: 10.3390/bios14080370.
Viral hepatitis is a systemic infectious diseases caused by various hepatitis viruses, primarily leading to liver damage. It is widely prevalent worldwide, with hepatitis viruses categorized into five types: hepatitis A, B, C, D, and E, based on their etiology. Currently, the detection of hepatitis viruses relies on methods such as enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy to observe and identify viral particles, and in situ hybridization to detect viral DNA in tissues. However, these methods have limitations, including low sensitivity, high error rates in results, and potential false negative reactions due to occult serum infection conditions. To address these challenges, we have designed an AuNPs-DNA walker method that uses gold nanoparticles (AuNPs) and complementary DNA strands for detecting viral DNA fragments through a colorimetric assay and fluorescence detection. The DNA walker, attached to gold nanoparticles, comprises a long walking strand with a probe sequence bound and stem-loop structural strands featuring a modified fluorescent molecule at the 3' end, which contains the DNAzyme structural domain. Upon the addition of virus fragments, the target sequence binds to the probe chains. Subsequently, the long walking strand is released and continuously hybridizes with the stem-loop structural strand. The DNAzyme undergoes hydrolytical cleavage by Mg, breaking the stem-loop structural strand into linear single strands. As a result of these structural changes, the negative charge density in the solution decreases, weakening spatial repulsion and rapidly reducing the stability of the DNA walker. This leads to aggregation upon the addition of a high-salt solution, accompanied by a color change. Virus typing can be performed through fluorescence detection. The innovative method can detect DNA/RNA fragments with high specificity for the target sequence, reaching concentrations as low as 1 nM. Overall, our approach offers a more convenient and reliable method for the detection of hepatitis viruses.
病毒性肝炎是一种由各种肝炎病毒引起的全身性传染病，主要导致肝脏损伤。它在全球广泛流行，根据病因，肝炎病毒分为甲型、乙型、丙型、丁型和戊型五种类型。目前，肝炎病毒的检测依赖于酶联免疫吸附测定（ELISA）、免疫电镜观察和识别病毒颗粒，以及原位杂交检测组织中的病毒 DNA 等方法。然而，这些方法存在局限性，包括灵敏度低、结果误差率高，以及由于隐匿性血清感染情况而导致潜在的假阴性反应。为了解决这些挑战，我们设计了一种 AuNPs-DNA walker 方法，该方法使用金纳米颗粒（AuNPs）和互补 DNA 链通过比色法和荧光检测来检测病毒 DNA 片段。与金纳米颗粒相连的 DNA walker 由一条带有探针序列的长行走链和一条带有修饰荧光分子的茎环结构链组成，该分子位于 3' 端，包含 DNA 酶结构域。加入病毒片段后，目标序列与探针链结合。随后，长行走链被释放并与茎环结构链持续杂交。DNA 酶在 Mg 的作用下发生水解切割，将茎环结构链断裂成线性单链。由于这些结构变化，溶液中的负电荷密度降低，空间排斥减弱，DNA walker 的稳定性迅速降低。当加入高盐溶液时，会发生聚集，同时伴随着颜色变化。通过荧光检测可以进行病毒分型。该创新方法对目标序列具有高度特异性，可以检测到低至 1 nM 的 DNA/RNA 片段。总的来说，我们的方法为肝炎病毒的检测提供了一种更方便、更可靠的方法。
