CHA-based dual signal amplification immunofluorescence biosensor for ultrasensitive detection of dimethomorph

Nucleic acids are considered as perfect programmable materials for cascade signal amplification and not merely as genetic information carriers. Among them, catalytic hairpin assembly (CHA), an enzyme-free, high-efficiency, and isothermal amplification method, is a typical example. A typical CHA reaction is initiated by single-stranded analytes, and substrate hairpins are successively opened, resulting in thermodynamically stable duplexes. CHA circuits, which were first proposed in 2008, present dozens of systems today. Through in-depth research on mechanisms, the CHA circuits have been continuously enriched with diverse reaction systems and improved analytical performance. After a short time, the CHA reaction can realize exponential amplification under isothermal conditions. Under certain conditions, the CHA reaction can even achieve 600 000-fold signal amplification. Owing to its promising versatility, CHA is able to be applied for analysis of various markers in vitro and in living cells. Also, CHA is integrated with nanomaterials and other molecular biotechnologies to produce diverse readouts. Herein, the varied CHA mechanisms, hairpin designs, and reaction conditions are introduced in detail. Additionally, biosensors based on CHA are presented. Finally, challenges and the outlook of CHA development are considered.

Human telomerase RNA (hTR), an important biomarker for cancer diagnosis, is the template for the synthesis of telomeric DNA repeats and is found to be 7-fold overexpressed in tumor cells. Herein, we present a photoelectrochemical (PEC) biosensor for hTR detection coupled with a novel amplification strategy based on cascades of catalytic hairpin assembly (CHA) and hyperbranched hybridization chain reaction (HB-HCR). At the electrode surface, thiolated hairpin 1 probes were immobilized on deposited CdS nanoparticles via a Cd-S bond. In the presence of target hTR, a CHA reaction was triggered and the exposing of trigger1 could further initiate an HB-HCR reaction to form abundant hemin/G-quadruplex DNAzymes containing dendritic DNA structure. The DNAzymes' catalytic precipitation of 4-chloro-1-naphthol (4-CN) by H2O2 subsequently took place on the surface of the PEC electrode and efficiently suppressed the photocurrent output. Therefore, the change of photocurrent response had a positive linear relationship with logarithmic value of hTR concentration varying from 200 fM to 20.0 nM with a limit of detection (LOD) of 17.0 fM. The LOD for CHA/HB-HCR was about 8.8-fold lower than that of CHA/linear-branched HCR (CHA/LB-HCR) and 547-fold lower than that of CHA. By coupling the feature of high signal amplification capacity for DNA nanotechnology, a prominently stable, reproducible, and selective PEC biosensor was successfully constructed and applied in hTR detection.

Fiber optic biosensor for detection of genetically modified food based on catalytic hairpin assembly reaction and nanocomposites assisted signal amplification

An ultrasensitive photochemical biosensor based on the target miRNA-triggered catalytic hairpin assembly (CHA) reaction between Au nanoparticles (AuNPs)/C3N4 nanosheets and CdS quantum dots (QDs) was developed for the determination of miRNAs. Firstly, AuNPs/C3N4 nanosheets were immobilized onto a working glassy carbon electrode. Then, the hairpin probe 1 (H1) was loaded through Au-S bonding. Afterward, the unbound sites were blocked with 6-mercaptohexanol to avoid nonspecific adsorption. In the presence of the target miRNA, the CHA reaction between the H1 and hairpin probe 2-CdS QDs (H2-CdS QDs) could be triggered. As a result, the AuNPs/C3N4 nanosheet and CdS QDs were linked by the double helix structure H1-H2. Unlike the other CHA reactions, H2 used in this work is longer than H1 so that the AuNPs/C3N4 nanosheets could touch the CdS QDs. Given the matched energy band positions between the C3N4 nanosheet and CdS QDs, a strong photocurrent could be obtained after the CHA reaction was triggered by the target miRNA. In addition, p-type C3N4 nanosheets and n-type CdS QDs presented reduction photocurrents and oxidation photocurrents, respectively. Therefore, the photocurrents were vectors in this design that can eliminate the interference of nonspecific adsorption and avoid the generation of false-positive signals. Under the optimal conditions, the limit of detection was 92 aM. The constructed photoelectrochemical biosensor showed good reproducibility and selectivity in the analysis of serum samples, which indicates its great prospects in disease diagnostics and bioanalysis.

An amplified electrochemical biosensing scheme is described for lead(II) ions. It is making use of DNAzyme-assisted target recycling and catalytic hairpin assembly (CHA). The hairpin strand (substrate probe for the Pb2+-based DNAzyme; referred to as SP) is composed of trigger probe (TP) and a capture probe 1 attached to gold nanoparticles (AuNP). In the presence of the enzyme probe that partially hybridizes with SP, the introduction of Pb2+ triggers target recycling and drives the highly amplified translation of target Pb(II) to TP. The CHA reaction is further initiated by TP. The modified AuNP act as the connecting unit, and this leads to the formation of a 3D DNA-AuNP network on the electrode (which is the third amplification step). It can bind the positively charged redox mediator RuHex via electrostatic interaction for electrochemical detection. This biosensor has a low detection limit (95 pM) and any analytical range that covers the 100 pM to 5μM Pb(II) concentration range. It is selective over other divalent metal ions. It was applied to the determination of Pb2+ in spiked real-world samples. Graphical abstract Schematic presentation of the electrochemical biosensor. The triply amplified electrochemical assay is based on the use of DNAzyme-assisted target recycling with catalytic hairpin assembly (CHA) reaction for sensitive and selective determination of lead ion (Pb2+). AuNP: gold nanoparticles; SP: substrate probe; EP: enzyme probe.

Early detection of human papillomavirus (HPV) is crucial for the treatment and prevention of some specific skin diseases and related cancers such as cervical cancer, anal cancer, penile cancer, etc. Herein, we designed a primer exchange reaction (PER)-based DNA cube captor (PDCC) to capture Raman molecular labeled DNA for high specificity and efficient detection of HPV. First, we converted the target into large amounts of single-stranded DNA based on PER. Second, the single-stranded DNA initiated catalytic hairpin assembly (CHA) reaction, facilitating the capture of Raman molecular labeled DNA onto the DNA cube nanostructures (DCNs). Then the DCNs were subsequently immobilized on gold hexagonal plates (GHPs) for Raman detection. Of note, the identification of the target by PER exhibits high specificity, as the PER reaction occurs exclusively when the target perfectly matches the PER hairpin. Moreover, the CHA reaction can occur on four sides of this structure, and the reaction efficiency is enhanced by the proximity effect. This work developed a device named PDCC that captured Raman molecular labeled DNA, which can realize highly sensitive detection of HPV-58. This DNA cube captor has a high reaction efficiency and specificity for HPV, which plays an indispensable role in diagnosis and treatment of related cancers.

Dual-mode corroborative detection of miRNA-21 based on catalytic hairpin assembly reaction and oxygen reduction reaction combined strategy for the signal amplification

Localized DNA catalytic hairpin assembly reaction on DNA origami for tumor-associated microRNA detection and imaging in live cells

A reproducible electrochemical biosensor for tobramycin highly sensitive detection based on ExoIII-assisted nucleic acid circulation and CHA reaction

MicroRNAs (miRNAs) play important roles in many biological processes and are associated with various diseases, especially cancers. Combination of technological developments such as nanomaterials, functional enzyme-mediated reactions, and DNA nanotechnology holds great potential for high-performance detection of miRNAs in molecular diagnostic systems. In this work, we have fabricated a cascade signal amplification platform through integrating duplex-specific nuclease (DSN)-assisted target recycling with catalytic hairpin assembly (CHA) reaction for the detection of microRNA-141 (miR-141). The target recycling process driven by DSN results in highly amplified translation of target miRNA to single-stranded connector DNA fragments. The CHA reaction is further initiated by connector DNAs using hairpin-modified gold nanoparticles (HP-AuNPs) as the sensing unit, leading to the formation of AuNP network architecture on the electrode for electrochemical and photoelectrochemical detection of miR-141 in signal-on and signal-off modes, respectively. The developed electrochemical biosensor exhibits a detection limit down to 25.1 aM miR-141 (60 copies in 4 μL sample) and excellent selectivity to discriminate a single base-mismatched sequence and other miRNAs. This assay is also applied to the determination of miR-141 in total RNAs extracted from human breast cancer cells (MDA-MB-231), confirming the applicability of this method for absolute quantification of specific miRNAs in real-world samples. Furthermore, two-input AND and INHIBIT (INH) logic gates are constructed to detect miRNAs. In particular, the AND gate achieves cell-specific gate activation based on expression profiles of miR-141 and microRNA-21 (miR-21). Therefore, our proposed cascade amplification platform has great potential applications in miRNA-related clinical diagnostics and biochemical research.

Using6-carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMRA) as fluorescent signals a ratiometric fluorescent three-dimensional (3D) DNA walker based on a catalytic hairpin assembly (CHA) reaction for microRNA-122 detectionwas constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. Thedual emission ratio fluorescent signal with a single excitation wavelength was usedas the signal output. This strategy combines DNA walker with CHA reaction and proportional fluorescence signal output methods, which can effectively reduce the background fluorescence signal and the risk of generating false-positive signals. Thus, the impact of environmental factors on the experimentis reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488nm and the maximum fluorescence emission wavelength of 520nm and 580nm, respectively. It has a good linear response at a microRNA concentration range of 156.0pM ~ 7.00nM and a detection limit of 42.94pM. This strategy has been successfully appliedto detect microRNAs in spiked serum samples. Graphical abstract Schematic representation of three-dimensional (3D) DNA walker constructed using catalytic hairpin self-assembly reaction (CHA)-assisted amplification and ratiometric fluorescence signal output for the detection of miRNA-122 closely related to hepatitis.

A highly sensitive surface-enhanced Raman scattering (SERS) biosensor has been developedfor the detection of microRNA-21 (miR-21) using an isothermal enzyme-free cascade amplification method involving catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The CHA reaction is triggered by the target miR-21, which causes hairpin DNA (C1 and C2) to self-assemble into CHA products. After AgNPs@Capture captures the resulting CHA product, the HCR reaction is started, forming long-stranded DNA on the surface of AgNPs. A strong SERS signal is generated due to the presence of a large amount of the Raman reporter methylene blue (MB) in the vicinity of the SERS "hot spot" on the surface of AgNPs. The monitoring of the SERS signal changes of MB allows for the highly sensitive and specific detection of miR-21. In optimal conditions, the biosensor exhibits a satisfactory linear range and a low detection limit for miR-21 of 42.3 fM. Additionally, this SERS biosensor shows outstanding selectivity and reproducibility. The application of this methodology to clinical blood samples allows for the differentiation of cancer patients from healthy controls. As a result, the CHA-HCR amplification strategy used in this SERS biosensor could be a useful tool for miRNA detection and early cancer screening.

Target-initiated DNA release-directed catalytic hairpin assembly-based ultrasensitive cyclic amplification sensor detection of serum miRNA

Electrochemical detection of microRNA based on SA-PPy/AuNPs nanocomposite with the signal amplification through catalytic hairpin assembly reaction and the spontaneous catalytic reaction of Fe3+/Cu2+

Cholesterol-modified DNA nanostructures enable a cascaded CHA-HCR system for enhanced miRNA detection in liquid biopsies.