Photoactive conjugated microporous polymer/carbon nanotube coupled with T-junction recycling dual-strand displacement amplification for sensing N-Gene of COVID-19

In this work, a novel conjugated microporous polymer on carbon nanotube composite (CMP-CNTs) was synthesized as photoelectrochemical (PEC) signal probe to construct sensitive PEC biosensor for sensing N-Gene of COVID-19 by integrating with an ingenious target-trigger T-junction recycling dual-strand displacement amplification (T-DSDA). The CMP-CNTs composites has an ideal photoelectrical conversion efficiency owing to the appearance of a good band matching that can effectively promote electron-hole pairs separation and accelerate carrier migration, thereby generating an extremely high initial photocurrent. Meanwhile, the T-DSDA with superior target conversion efficiency to traditional approaches could convert the small number of targets into extensive output DNAs, leading to the in-situ generation of quench agent N-GQDS decorated nanowires on electrode for significantly reducing initial photocurrent. The results demonstrated that proposed PEC biosensor had a high sensitivity towards N-Gene of COVID-19 and the detection limit was 33 aM, which provided a new way to build the simple, sensitive, and reliable sensing platform for great potential in biological analysis and early clinical diagnosis. [Display omitted] • New CMP-CNTs composites have a good energy band matching, which can boost visible light absorption efficiency. • A target-trigger T-junction dual-strand displacement amplification transform small targets into massive output DNAs. • Output DNAs trigger HCR to generate N-GQDs modified nanowires in situ, which significantly reduces the PEC signal. • The proposed PEC biosensor of CMP-CNTs composites achieved ultra-sensitive detection of N-Gene of COVID-19. [ FROM AUTHOR]

Photoactive Conjugated Microporous Polymer/Carbon Nanotube Coupled with T-junction Recycling Dual-Strand Displacement Amplification for Sensing N-Gene of COVID-19

In this work, a novel conjugated microporous polymer on carbon nanotube composite (CMP-CNTs) was synthesized as photoelectrochemical (PEC) signal probe to construct sensitive PEC biosensor for sensing N-Gene of COVID-19 by integrating with an ingenious target-trigger T-junction recycling dual-strand displacement amplification (T-DSDA). The CMP-CNTs composites has an ideal photoelectrical conversion efficiency owing to the appearance of a good band matching that can effectively promote electron-hole pairs separation and accelerate carrier migration, thereby generating an extremely high initial photocurrent. Meanwhile, the T-DSDA with superior target conversion efficiency to traditional approaches could convert the small number of targets into extensive output DNAs, leading to the in-situ generation of quench agent N-GQDS decorated nanowires on electrode for significantly reducing initial photocurrent. The results demonstrated that proposed PEC biosensor had a high sensitivity towards N-Gene of COVID-19 and the detection limit was 33 aM, which provided a new way to build the simple, sensitive, and reliable sensing platform for great potential in biological analysis and early clinical diagnosis.

Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription.

Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2 fM and 200 fM within 1 h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.

DNA Hairpins and Dumbbell-Wheel Transitions Amplified Walking Nanomachine for Ultrasensitive Nucleic Acid Detection.

Nucleic acids, including circulating tumor DNA (ctDNA), microRNA, and virus DNA/RNA, have been widely applied as potential disease biomarkers for early clinical diagnosis. In this study, we present a concept of DNA nanostructures transitions for the construction of DNA bipedal walking nanomachine, which integrates dual signal amplification for direct nucleic acid assay. DNA hairpins transition is developed to facilitate the generation of multiple target sequences; meanwhile, the subsequent DNA dumbbell-wheel transition is controlled to achieve the bipedal walker, which cleaves multiple tracks around electrode surface. Through combination of strand displacement reaction and digestion cycles, DNA monolayer at the electrode interface could be engineered and target-induced signal variation is realized. In addition, pH-assisted detachable intermolecular DNA triplex design is utilized for the regeneration of electrochemical biosensor. The high consistency between this work and standard quantitative polymerase chain reaction is validated. Moreover, the feasibilities of this biosensor to detect ctDNA and SARS-CoV-2 RNA in clinical samples are demonstrated with satisfactory accuracy and reliability. Therefore, the proposed approach has great potential applications for nucleic acid based clinical diagnostics.

Aligner-Mediated Cleavage-Based Isothermal Amplification for SARS-CoV-2 RNA Detection.

Rapid detection of SARS-CoV-2 RNA is critical for reducing the global transmission of COVID-19. Here, we report a simple and versatile assay for detection of SARS-CoV-2 RNA based on aligner-mediated cleavage-based strand displacement amplification (AMC-SDA). The entire amplification procedure takes less than 25 min without professional instruments or requirement of specific target sequences and can reach a limit of detection of attomolar RNA concentration. Using pseudovirus as mimicry of clinical SARS-CoV-2 positive samples, we achieved a diagnostic accuracy of 100% in 10 simulated samples (five positive and five negative). We anticipate that our method will provide a universal platform for rapid and accurate detection of emerging infectious diseases.