Dual-cycle Amplification Research Articles

Immobilization coupling with aptamer assisted dual cycle amplification for sensitive sEVs isolation and analysis.

Precise identification of small extracellular vesicles (sEVs) is crucial for improving disease diagnosis and treatments, such as bladder cancer. However, accurate isolation and simultaneously quantification of sEVs remain a huge challenge. We have introduced a new technique that combines immobilization with aptamer-assisted dual cycle amplification to isolate and analyze sEVs with high sensitivity. In this method, the CD9 protein antibody is attached to the plate's surface for the initial identification of sEVs, while an aptamer probe is used to detect the exosomal surface protein CD63. We have created an sEVs-surface method that combines target recognition initiated signal recycling and rolling circle amplification (RCA) for signal amplification. This approach allows for the "AND" logic analysis of dual biomarkers, enabling both sEVs quantification and tracing. The proposed approach has a broad detection range and a low limit of detection. Moreover, the established method showed good stability in detecting sEVs with a low coefficient of variation. Our method can effectively isolate certain sEVs and accurately identify them, making it suitable for many uses in biological science, biomedical engineering, and personalized medicine.

Highly sensitive miRNA detection of early-stage laryngeal carcinoma using a solid-state Au nanocone arrays fabricated LoC-SERS analysis system coupled with target-triggered dual cycle amplification strategy

Herein, with a target-triggered dual cycle amplification strategy, a novel lab-on-a-chip-based surface-enhanced Raman scattering (LoC-SERS) analysis system was established to analyze miRNAs quantitatively. Due to a more substantial complementary pairing effect, the presence of targets can replace the single-stranded DNA S1 (S1) from the single-stranded DNA F1 (F1) and single-stranded DNA S2 (S2) can replace and release the targets similarly. Thus, target can cyclically trigger the cycle Ⅰ and release S1 continuously. Cycle Ⅱ is the catalyzed hairpin assembly (CHA) event between the hairpin DNA 1 and hairpin DNA 2, which can connect the Ag-Au nanorods (Ag-AuNRs) to the surface of solid-state Au nanocone arrays (AuNCAs). Owing the dual cycle amplification strategy, localized surface plasmon resonance (LSPR) can be excited and significantly magnify the local electromagnetic field for SERS signal enhancement. Thus, ultrasensitive detection of miR-21 and miR-106b was achieved with the limit of detection as low as aM level. Attributed to the hydrophilic treatment and the design of capillary pump, the detection can be finished without any external pumps. Moreover, the system exhibited good practicability in the analysis of real serum samples obtained from Laryngeal carcinoma (LC) patients and healthy subjects. Therefore, the system is a promising candidate in clinical application and exhibited potential for the prediction, diagnosis, monitoring, and staging of LC.

A dual-cycle amplification-based electrochemical platform for sensitive detection of tobramycin

BackgroundTobramycin (TOB), an essential aminoglycoside antibiotic in human life, poses potential threats due to its residues in the environment. The primary concern is the adverse impact of excessive TOB on human kidneys, hearing, and other organs, significantly affecting human health. Constructing a sensitive electrochemical platform for simple and rapid trace detection is crucial. Herein, to enhance the sensitivity of TOB detection in the environment and mitigate the risks associated with residual antibiotics, an ultrasensitive electrochemical aptasensor was developed. ResultsThe sensor employs a dual-cycle amplification strategy involving catalytic hairpin assembly (CHA) and exonuclease III (Exo III) for efficient signal amplification. Simultaneously, the electrode performance was optimized by incorporating gold nanowires (AuNWs) onto the surface of reduced graphene oxide (PDA-rGO). Specifically, in the presence of TOB, which binds to the aptamer (Apt), dsDNA dissociates, releasing cDNA to open hairpin 1 (HP1) and initiate the CHA cycle with the participation of hairpin 2 (HP2). Exo III shears HP1 in the HP1/HP2 complex, freeing HP2 to participate in the CHA cycle again. Ultimately, a significant amount of signal label is retained on the electrode by hybridizing with sheared HP1, generating a robust electrical signal. SignificanceThrough the signal amplification strategy, the aptasensor design provides a broad linear range of 0.005–500 nM, with a low detection limit of 0.112 pM for TOB. It is worth mentioning that the aptasensor displayed favorable stability, specificity, and reproducibility, and has been successfully applied to practical samples, demonstrating its utility in practical applications.

Ultrasensitive quantitation of Paraquat based on a small molecule-induced dual-cycle amplification strategy

Paraquat (PQ) is a typical biotoxic small molecule. Knowledge of how to directly introduce it into cyclic amplification rather than transform it into a secondary target is lacking in current analytical methods. Considering the urgent need for trace pesticide residue detection and the inherent defects of small molecule analysis, a CRISPR/Cas12a-driven small molecule-induced dual-cycle strategy was developed based on the immune competition method. The key to signal amplification is the mutual activation and acceleration between Cycle 1 triggered by the small molecule and Cycle 2 driven by CRISPR/Cas12a. Impressively, small molecules have been successfully incorporated into the dual-cycle strategy, which achieves a low detection limit (3.1 pg/mL) and a wide linear range (from 10 pg/mL to 50 μg/mL). Moreover, the designed biosensor was successfully employed to evaluate the PQ residual level in real samples and showed effective implementation for the bioanalysis of small molecule targets and pesticide residue-related food safety.

A dual-amplified ROS-responsive nanosystem with self-accelerating drug release for synergistic chemotherapy.

In this work, we have developed a tumor-specific self-accelerating prodrug activation nanosystem consisting of self-amplifying degradable polyprodrug PEG-TA-CA-DOX and encapsulated fluorescent prodrug BCyNH2, equipped with a reactive oxygen species dual-cycle amplification effect. Furthermore, activated CyNH2 is a therapeutic agent with potential to synergistically improve chemotherapy.

Allosteric Probe-Initiated Wash-Free Method for Sensitive Extracellular Vesicle Detection through Dual Cycle-Assisted CRISPR-Cas12a.

Extracellular vesicles (EVs) are emerging as promising biomarkers for cancer diagnosis and therapy. Recognizing low-abundance EVs from clinical samples in an easy-to-operate way is highly desired but remains a challenge. Herein, we established an allosteric probe-initiated dual cycle amplification-assisted CRISPR-Cas12a (AID-Cas) platform for sensitive detection of EVs in a wash-free way. In AID-Cas, the allosteric probe can specifically recognize and bind with target EVs and thus initiate the following dual-cycle amplification. Subsequently, the amplified products were transcribed to generate numerous single-stranded RNAs, which could work as crRNA to trigger the trans-cleavage of CRISPR-Cas12a. Consequently, the proposed approach achieved a good linear response to extracted EVs in a concentration range from 102 to 106 particles/μL. Because of its high sensitivity, together with its wash-free convenience, the proposed strategy could have promising clinical potentials for early diagnosis of cancers.

Hg2+ Electrochemical Biosensor Based on Target Triggered Exonuclease III-Assisted Dual Cycle Amplification and Tetrahedron DNA Nanostructures as Signal Molecule Carrier

A novel Hg2+ electrochemical assay was presented based on target triggered exonuclease III (Exo III) assisted dual cycle amplification and tetrahedron DNA (TDNA) nanostructures as efficient signal molecules carrier. The thymine bases rich (T-rich) hairpin 1 (HP1) was wisely designed with the significant report DNA (RDNA) sequence in the loop, which could be acted as probes for specially recognizing Hg2+ with formation of duplex-like structure through T-Hg2+-T coordination. This duplex-like structure could be digested by Exo III to release numerous Hg2+ for next cycle, accompanying with the generation of an important digestion product RDNA for opening hairpin 2 (HP2) on electrode. After the introduction of hairpin 3 (HP3) on electrode via strand displacement reactions, the RDNA could be released again for next cycle, leading to the assembly of large amounts of HP3 for decorating TDNA. Finally, by using the TDNA as carriers for loading substantial signal probe thionine (Thi), a remarkable electrochemical signal could be acquired for quantitative detection of target. As a result, the developed biosensor showed a low detection limit of 33 fM for Hg2+ determination. This strategy designed a novel electrochemical biosensor, which offered a new way for metal ion detection, showing potential applications in environmental monitoring.

Dual cycle amplification and dual signal enhancement assisted sensitive SERS assay of MicroRNA

A sensitive surface-enhanced Raman scattering (SERS) approach has been developed for detection of microRNA (miRNA) based on target-triggered dual signal amplification including strand displancement amplification (SDA) and hybridization chain reaction (HCR). With the assistant of polymerase and nicking endonuclease (NEase), target miRNA combines with the single stranded template DNA to generate a great amount of trigger DNA which can induce HCR. Coupled the dual cycle amplification of SDA and HCR with the dual enhancement of gold nanoparticles (AuNPs), a low detection limit of 0.5 fM for miRNA is obtained using the proposed strategy. With high sensitivity, universality, rapid analysis, and high selectivity, this method has a great potential for detecting biomolecules with trace amounts in bioanalysis and clinical biomedicine.

A simple and rapid dual-cycle amplification strategy for microRNA based on graphene oxide and exonuclease III-assisted fluorescence recovery

A simple microRNA detection method by combining Graphene Oxide (GO) fluorescence quenching with exonuclease III (Exo-III) aided cycling amplification was developed.

TTE DNA-Cu NPs: enhanced fluorescence and application in a target DNA triggered dual-cycle amplification biosensor.

A crowded TTE DNA structure for the preparation of Cu NPs with enhanced fluorescence was prepared and applied for the ultrasensitive detection of target DNA.