病毒性肝炎是由各种肝炎病毒引起的全身性传染病,主要导致肝脏损伤。它在世界范围内广泛流行,肝炎病毒分为五种类型:甲型肝炎,B,C,D,E,根据他们的病因。目前,肝炎病毒的检测依赖于酶联免疫吸附测定(ELISA)等方法,免疫电子显微镜观察和鉴定病毒颗粒,和原位杂交检测组织中的病毒DNA。然而,这些方法有局限性,包括低灵敏度,结果错误率高,和潜在的假阴性反应由于隐匿性血清感染条件。为了应对这些挑战,我们设计了一种AuNPs-DNAWalker方法,该方法使用金纳米颗粒(AuNPs)和互补DNA链通过比色测定和荧光检测来检测病毒DNA片段。那个DNA步行者,附着在金纳米粒子上,包括一个长的步行链与探针序列结合和茎环结构链的特点是在3'端修饰的荧光分子,其中包含DNAzyme结构域。添加病毒片段后,靶序列与探针链结合。随后,长行走链被释放并与茎环结构链连续杂交。DNAzyme经历Mg2的水解裂解,将茎环结构链分解成线性单链。由于这些结构性变化,溶液中的负电荷密度降低,削弱空间斥力,迅速降低DNA助行器的稳定性。这导致在添加高盐溶液时聚集,伴随着颜色的变化。病毒分型可以通过荧光检测进行。该方法能对靶序列进行高特异性的DNA/RNA片段检测,达到低至1nM的浓度。总的来说,我们的方法为肝炎病毒的检测提供了更方便和可靠的方法。
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 Mg2+, 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.