基于卟啉仿生纳米探针的电致化学发光核酸传感器的研究

DNA assay has already taken a key place in molecular diagnosis, genetic therapy, etc. Ultrasensitive detection method for nucleotide could realize the determination of DNA/RNA at exceptionally low level, of which the enzyme-linked signal amplification protocol prevailed. However, the bioactivity, low tagging rate and unstability of natural enzymes held it back. In view of the metalloporphyrins as cofactor of several protein enzymes, this project proposes a novel strategy for nucleotide assay by integrating the orderly nanoassembly of porphyrins, nanoscale signal amplification and quantum dots (QDs)-based electrochemiluminescence (ECL) technology. The interaction via axial ligation between porphyrins and graphene-like nanomaterials is employed to fabricate the biomimetic nanoprobe, which would possess mimicking catalytic activity and biorecognition specificity by biocoupling reaction. Meanwhile, sequcences of peptide nucleic acid are employed as the capture probes against nonspecific adsorption of tagging molecules. The ultrasensitve QDs-based ECL DNA assays can thus be achieved by coupling multiple quenching effects of the probe and its catalytic products on ECL emission with versatile DNA-based signal amplification techniques. Various spectroscopic and morphological characterizations are applied for stepwise real-time monitoring the construction of detection systems. By taking resort to molecular modeling and computational chemistry, intrinsic biocatalytic mechanisms concerned about the interactions could be investigated to reveal the synergistic effect of each individual component. Furthermore, the optimization of preparation and detection parameters would promise a nice sensing performance. The proposed system will be practically utilized to detect specific cancer-related protein-expressing genes, which would not only lay a foundation for clinic diagnoses; but provide novel, efficient, facile and universal enzymatic substitutes for nano-biosensing.

核酸分析已在分子诊断、基因治疗等领域发挥重要作用，高灵敏核酸检测方法可实现极低浓度DNA/RNA分析，这其中以酶联信号放大技术尤为突出。该技术目前受制于天然酶标的生物活性、低标记率和储备不稳定性。鉴于许多蛋白酶以金属卟啉为辅基，本项目提出了一种融合卟啉有序纳米组装、纳米信号放大和量子点电致化学发光(ECL)技术为一体的核酸分析新策略。该策略通过卟啉与类石墨烯纳米材料的轴向配位作用构建新颖的仿生纳米探针，通过生物偶联而兼具模拟酶催化活性和生物识别特异性；采用肽核酸等新型靶标，耦合探针及其催化产物对ECL辐射的多重猝灭效应和灵活多样的DNA信号放大技术，实现高灵敏核酸分析；并联用多种先进表征手段探索仿生催化机制，揭示组分间协同效应，继而优化参数以获得最佳分析性能。最终将所设计的体系用于表达癌症相关蛋白特定基因序列的分析，为临床诊断奠定基础，并为纳米生物传感提供新颖、高效、便利且普适的酶替代物。

过氧化物酶的人工合成是目前酶工程研究的重点，其高质的批量生产及下游有效的化学修饰将有助于推动新药开发、临床诊断、环境监测、司法鉴定、军事防护和食品安全等多个相关行业和产业的发展。然而，过氧化物酶的生物制备效率低，须复杂的提取、分离和纯化工艺，成本昂贵，储存和反应条件比较苛刻且极易失活，这些都极大制约其衍生的生物产品的民用普及。为克服上述缺陷，本项目提出了一种新颖的融合纳米科技的过氧化物酶模拟加工工艺。其基本思路是从结构和分子生物学层面，“自底向上”的构建酶，技术关键在于天然酶结构的解构与仿生重建，即提取过氧化物酶的酶促反应活性中心−卟啉，以廉价均质、具有类石墨烯结构的二维纳米氮化碳单层材料作为载体逼近并还原该辅基的化学环境，通过简易的取代基修饰、π−π共轭或轴向配位的组装方式，实现在结构上对天然酶的模拟；并进一步以酶促反应涉及的若干动力学参数，如米氏常数(KM)、转换数(TN)、催化常数(kcat)、催化效率等作为主要技术指标，通过联用多种先进的表征分析手段和Lineweaver−Burk双倒数作图法，比较并验证所得模拟酶产品在活性上具备与天然酶相当的竞争力及其对温度、离子强度、溶液pH值等反应、储备条件的敏感度和耐受力。进一步用生物偶联技术赋予模拟酶识别特异性，通过开发配套的基因分析试剂盒，构建实际的病毒基因检测传感器件并记录信号的灵敏度和准确性，以考察模拟酶的生物标记率和相容性，从而证实其在生物医学领域的应用价值和对天然酶的替代潜力。