Abstract:

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 GCD-Bea 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.

Key words: Fluorescent carbon dots, Guanidine modification, Nucleic acid testing, Novel coronavirus, High specificity, High sensitivity, Point of care testing