Kalsoom Iqra, Shehzadi Kiran, Irfan Muhammad, Qiu Lili, Wang Yifei, Xu Zhibin, Meng Zihui
Beijing Institute of Technology School of Chemistry and Chemical Engineering, China.
Beijing Institute of Technology School of Chemistry and Chemical Engineering, China.
Biosens Bioelectron. 2025 Apr 1;273:117091. doi: 10.1016/j.bios.2024.117091. Epub 2025 Jan 8.
Photonic crystal-based aptasensors for viral proteins detection offer the advantage of producing visible readouts. However, they usually suffer from limited sensitivity and high non-specific background noise. A significant contributing factor to these issues is the use of fixed-conformation aptamers in these sensors. To address this challenge, we have developed an approach to transforming aptamers into molecular switches that undergo conformational changes upon target binding. This strategy enhances the performance of colorimetric aptasensors by improving sensitivity and reducing background noise without the need for post-SELEX modifications. In this study, we present an efficient aptamer screening method designed to facilitate the development of target-responsive aptamer structure switches from pre-existing aptamers with stable conformations. A novel algorithm was developed that integrates binding motif information with secondary structure data to generate three ssDNA libraries. Through virtual screening, we identified an aptamer with structure-switching properties (MSA-Apt-16) (Kd = 7.6 nM), and its structure-switching property was further validated using molecular simulations and circular dichroism spectroscopy. Further, the binding kinetics and selectivity of both aptamers were evaluated using SPR. MSA-Apt-16 displayed a kon of 1.39 × 10 Ms and a koff of 1.76 × 10 s, signifying a high binding affinity and selectivity. In contrast, Apt-H8 showed a kon of 1.22 × 10 Ms and a koff of 1.89 × 10s, showing a low binding affinity with RSV-G. Moreover, we demonstrated the enhanced sensitivity of the newly designed molecular switching aptamer (MSA-Apt-16) compared to the parent aptamer (Apt-H8) by incorporating them into the photonic crystal-based colorimetric sensing platform for the detection of RSV-G protein. The molecular switching aptasensor successfully detected RSV-G protein, exhibiting efficient color changes, significantly reduced background noise, and a limit of detection (LOD) of 1 pg/mL with a linear range of 1-500 pg/mL (signal-to-noise ratio = 3, R = 0.9489), and an analytical recovery of 94.0 ± 1.5 % to 102.0 ± 1.5 % in artificial serum, saliva, and nasal swab samples. In contrast, the parent aptamer demonstrated a LOD of 1 ng/mL with a linear range of 1-500 ng/mL (R = 0.9129). Our pipeline offers a robust and broadly applicable technique for developing aptamer switches personalized to specific targets, significantly reducing background noise and enhancing aptasensor efficiency, thus expanding the potential applications of aptasensors in various diagnostic fields.
用于病毒蛋白检测的基于光子晶体的适体传感器具有产生可见读数的优势。然而，它们通常灵敏度有限且非特异性背景噪声高。这些问题的一个重要促成因素是在这些传感器中使用固定构象的适体。为应对这一挑战，我们开发了一种将适体转化为分子开关的方法，该分子开关在与靶标结合时会发生构象变化。这种策略通过提高灵敏度和降低背景噪声来增强比色适体传感器的性能，而无需进行SELEX后修饰。在本研究中，我们提出了一种高效的适体筛选方法，旨在促进从具有稳定构象的现有适体开发靶标响应性适体结构开关。开发了一种新算法，该算法将结合基序信息与二级结构数据整合以生成三个单链DNA文库。通过虚拟筛选，我们鉴定出一种具有结构切换特性的适体（MSA-Apt-16）（解离常数Kd = 7.6 nM），并使用分子模拟和圆二色光谱进一步验证了其结构切换特性。此外，使用表面等离子体共振（SPR）评估了两种适体的结合动力学和选择性。MSA-Apt-16的结合速率常数kon为1.39×10⁶ M⁻¹s⁻¹，解离速率常数koff为1.76×10⁻³ s⁻¹，表明具有高结合亲和力和选择性。相比之下，Apt-H8的kon为1.22×10⁶ M⁻¹s⁻¹，koff为1.89×10⁻² s⁻¹，显示出与呼吸道合胞病毒糖蛋白（RSV-G）的结合亲和力较低。此外，通过将新设计的分子开关适体（MSA-Apt-16）和母体适体（Apt-H8）整合到基于光子晶体的比色传感平台中用于检测RSV-G蛋白，我们证明了新设计的分子开关适体相对于母体适体具有更高的灵敏度。分子开关适体传感器成功检测到RSV-G蛋白，表现出有效的颜色变化、显著降低的背景噪声，检测限（LOD）为1 pg/mL，线性范围为1-500 pg/mL（信噪比=3，R² = 0.9489），在人工血清、唾液和鼻拭子样本中的分析回收率为94.0±1.5%至102.0±1.5%。相比之下，母体适体的检测限为1 ng/mL，线性范围为1-500 ng/mL（R² = 0.9129）。我们的流程提供了一种强大且广泛适用的技术，用于开发针对特定靶标的个性化适体开关，显著降低背景噪声并提高适体传感器效率，从而扩大了适体传感器在各种诊断领域的潜在应用。
