用于高灵敏快速核酸检测的荧光碳点

Fluorescent Carbon Dots for Rapid and Highly Sensitive Detection of Nucleic Acids

逆转录-实时荧光定量聚合酶链式反应(RT-qPCR)已经广泛应用于核酸检测。然而,如何实现准确、快速即时检测仍然是亟待解决的关键问题。本研究设计了一种使用胍基修饰的荧光碳点(GCDs),用于高灵敏度以及高特异性的快速核酸检测。当GCDs与分子信标(Beacon)通过氢键作用结合后,GCDs的荧光被Beacon的荧光基团淬灭。当遇到目标核酸(Target DNA)之后,Beacon与Target DNA进行碱基互补配对,GCDs与Beacon脱离,GCDs自身荧光恢复。基于这种灵敏的荧光“off-on”,可实现在5 min内快速准确核酸检测,并可检测到体系内0.005 fmol∙L^(−1)(约300 copys∙mL^(−1))的核酸序列,相比于RT-qPCR所需要的2–4 h,该方法不需要进行核酸扩增,缩短了检测时间,有望为目前的核酸检测提供一个便捷有效的方法。

Antigen tests and nucleic acid detection via the reverse transcription quantitative polymerase chain reaction(RT-qPCR)have been widely used amid the spread of the new coronavirus disease(COVID-19).Despite its superior detection performance,RT-qPCR requires long detection times,expensive professional equipment,and detection personnel.By contrast,antigen tests can produce results within 15 min but often lack in terms of specificity and sensitivity.Therefore,the realization of accurate and rapid detection remains a crucial challenge.In this study,guanidine-modified fluorescent carbon dots(GCDs)were synthesized via a hydrothermal method using o-phenylenediamine and arginine as precursors for the rapid,highly sensitive,and specific detection of nucleic acids.The crude CDs were purified using combined silica gel and neutral alumina column chromatographies until yellow fluorescent GCDs with guanidine-modified edges were obtained.Notably,the yellow fluorescence of the GCDs,with a quantum yield of 9.8%,represents the main detection principle of this study.After incubating the GCDs with a molecular beacon(Bea)for 15 min to create hydrogen-bonded GCD-Bea pairs,a transfer of fluorescence resonance energy was initiated between the GCDs and the ROX fluorescence groups carried by the Bea.During this process,GCDs fluorescence was quenched,thus weakening the fluorescence of the GCD-Bea pairs.When the GCD-Bea pairs encountered target DNA molecules,the Beas and target DNAs underwent base complementary pairing,causing the GCDs and Bea to detach;the latter recovered the self-fluorescence of the GCDs,enabling qualitative detection of the target DNAs in the system.Fluorescence analyses revealed that the fluorescence of the target DNA group was enhanced by more than 20%compared with that of the control group.The entire fluorescence"off-on"DNA detection process described above was accomplished within 5 min,achieving a specificity of 95.45%.Furthermore,the lowest DNA detection concentration in the system was 0.005 fmol∙L^(−1)(approximately 300 copys∙mL^(−1)),and no acid amplification process was required.More importantly,after replacing the Bea sequence with the DNA sequences of other viruses or diseases,the obtained GCD-Bea pairs could still detect the corresponding target DNAs,confirming their capability of identifying target DNA sequences in a mixed system without the need for nucleic acid extraction.Additionally,compared with the 2–4 h typically required by qPCR,our GCDBea system could achieve considerably shortened detection times while also maintaining high specificity and sensitivity after Bea sequence replacements.Collectively,these characteristics are expected to provide a convenient and effective method for the detection of multiple viruses or diseases.