School of Tropical Medicine and Laboratory Medicine, Key Laboratory of Emergency and Trauma of Ministry of Education, Hainan Medical University, Haikou 571199, China.
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Anal Chem. 2021 Jul 20;93(28):9933-9938. doi: 10.1021/acs.analchem.1c02229. Epub 2021 Jul 6.
Sensitive detection of the SARS-CoV-2 protein remains a great research interest in clinical screening and diagnosis owing to the coronavirus epidemic. Here, an ultrasensitive chemiluminescence (CL) imaging strategy was developed through proximity hybridization to trigger the formation of a rolling circle-amplified G-quadruplex/hemin DNAzyme for the detection of the SARS-CoV-2 protein. The target protein was first recognized by a pair of DNA-antibody conjugates, Ab-1 and Ab-2, to form a proximity-ligated complex, Ab-1/SARS-CoV-2/Ab-2, which contained a DNA sequence complemental to block DNA and thus induced a strand displacement reaction to release the primer from a block/primer complex. The released primer then triggered a rolling circle amplification to form abundant DNAzyme units in the presence of hemin, which produced a strong chemiluminescent signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide. The proposed assay showed a detectable concentration range over 5 orders of magnitude with the detection limit down to 6.46 fg/mL. The excellent selectivity, simple procedure, acceptable accuracy, and intrinsic high throughput of the imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis.
由于冠状病毒的流行，对 SARS-CoV-2 蛋白的敏感检测仍然是临床筛选和诊断的重要研究课题。在这里，通过邻近杂交开发了一种超灵敏的化学发光（CL）成像策略，以触发滚环扩增 G-四链体/血红素 DNA 酶的形成，用于检测 SARS-CoV-2 蛋白。目标蛋白首先被一对 DNA-抗体偶联物 Ab-1 和 Ab-2 识别，形成邻近连接复合物 Ab-1/SARS-CoV-2/Ab-2，其中包含与阻断 DNA 互补的 DNA 序列，从而诱导链置换反应以从阻断/引物复合物中释放引物。释放的引物随后在血红素存在下触发滚环扩增，在该扩增过程中形成大量的 DNA 酶单元，通过催化过氧化氢对鲁米诺的氧化产生强化学发光信号，从而用于检测目标蛋白。所提出的测定法显示出可检测浓度范围超过 5 个数量级，检测限低至 6.46 fg/mL。该成像技术用于血清样本分析的出色选择性、简单的程序、可接受的准确性和内在的高通量，证明了所提出的检测方法在临床筛选和诊断中的潜在适用性。
