An ultrasensitive electrochemical biosensor with dual-amplification mode and enzyme-deposited silver for detection of miR-205-5p.

An electrochemical biosensor based on dual-amplified nucleic acid mode and biocatalytic silver deposition was constructed using catalytic hairpin assembly-hybrid chain reaction (CHA-HCR). The electrochemical detection of silver on the electrode by linear sweep voltammetry (LSV) can be utilized to quantitatively measure miR-205-5p since the amount of silver deposited on the electrode is proportional to the target nucleic acid. The current response values exhibit strong linearity with the logarithm of miR-205-5p concentrations ranging from 0.1 pM to 10 µM, and the detection limit is 28 fM. A consistent trend was found in the results of the qRT-PCR and electrochemical biosensor techniques, which were employed to determine the total RNA recovered from cells, respectively. Moreover, the constructed sensor was used to assess miR-205-5p on various cell counts, and the outcomes demonstrated the excellent analytical efficiency of the proposed strategy. The recoveries ranged from 97.85% to 115.3% with RSDs of 2.251% to 4.869% in human serum samples. Our electrochemical biosensor for miR-205-5p detection exhibits good specificity, high sensitivity, repeatability, and stability. It is a potentially useful sensing platform for tumor diagnosis and tumor type identification in clinical settings.

Highly sensitive and reliable detection of microRNA for clinically disease surveillance using SERS biosensor integrated with catalytic hairpin assembly amplification technology.

MicroRNAs (miRNAs) play an important regulatory role in several diseases, especially as a class of promising biomarkers for cancer diagnosis and prognosis. Here, a biosensor based on surface enhanced Raman spectroscopy (SERS) combined with catalytic hairpin assembly (CHA) amplification technology was developed for ultra-sensitive detection of miRNA-21 and miRNA-155 in breast cancer serum. By using CHA strategy, the extremely low concentration of target microRNA in human serum can be significantly amplified through the re-hybridization with thousands of hairpin probes to trigger amplification cycles. Besides, a sandwich SERS sensing chip with numerous hot spots and signal self-calibration was built through the linkage between two-dimensional Au-Si substrate and upper Ag@4-MBA@Au core-shell nanoparticles. Using this specially-designed biosensing platform, a low detection limit of 0.398 fM and 0.215 fM with a dynamic range from 1 fM to 10 nM can be achieved for the detection of miRNA-21 and miRNA-155, respectively. Additionally, the analysis of these two miRNAs in serum samples is capable of identifying the breast cancer subjects from normal ones with 100% of accuracy, as well as potentially evaluating the molecular types and prognosis for breast cancer. These results demonstrate that the proposed SERS with CHA technology would be an alternative method for highly sensitive and reliable detection of miRNA biomarkers contributing to breast cancer diagnosis and prognosis.

Ratiometric fluorescent 3D DNA walker and catalyzed hairpin assembly for determination of microRNA.

Using 6-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 detection was constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. The dual emission ratio fluorescent signal with a single excitation wavelength was used as 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 experiment is reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488 nm and the maximum fluorescence emission wavelength of 520 nm and 580 nm, respectively. It has a good linear response at a microRNA concentration range of 156.0 pM ~ 7.00 nM and a detection limit of 42.94 pM. This strategy has been successfully applied to 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.

l-DNA-Based Catalytic Hairpin Assembly Circuit.

Isothermal, enzyme-free amplification methods based on DNA strand-displacement reactions show great promise for applications in biosensing and disease diagnostics but operating such systems within biological environments remains extremely challenging due to the susceptibility of DNA to nuclease degradation. Here, we report a catalytic hairpin assembly (CHA) circuit constructed from nuclease-resistant l-DNA that is capable of unimpeded signal amplification in the presence of 10% fetal bovine serum (FBS). The superior biostability of the l-DNA CHA circuit relative to its native d-DNA counterpart was clearly demonstrated through a direct comparison of the two systems (d versus l) under various conditions. Importantly, we show that the l-CHA circuit can be sequence-specifically interfaced with an endogenous d-nucleic acid biomarker via an achiral peptide nucleic acid (PNA) intermediary, enabling catalytic detection of the target in FBS. Overall, this work establishes a blueprint for the detection of low-abundance nucleic acids in harsh biological environments and provides further impetus for the construction of DNA nanotechnology using l-oligonucleotides.

A coumarin-appended cyclometalated iridium(III) complex for visible light driven photoelectrochemical bioanalysis.

In this study, a coumarin-appended cyclometalated iridium (III) complex was prepared and demonstrated to be an efficient photoelectrochemical (PEC) active species with stable and reproducible cathodic photocurrent illuminated by visible light. A gold nanoparticles (AuNPs)-based PEC probe was assembled using the as-prepared iridium (III) complex as signal reporter. Integrating aptamer/protein proximity binding-triggered strand displacement and catalytic hairpin assembly (CHA) amplification strategy, an enzyme-free and sensitive PEC assay was developed. Benefiting from superior photon-to-current conversion character of the iridium (III) complex and effective amplification strategy, the proposed assay exhibited enhanced sensitivity for thrombin detection with a detection limit of 23 fM. It also showed a high specificity in serum samples. This study further demonstrated that cyclometalated iridium (III) complexes could be adopted as favorable photoactive material for bioanalysis by improving their ability of absorbance in the visible region.

Applications of Catalytic Hairpin Assembly Reaction in Biosensing.

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.

An enzyme-free electrochemical biosensor combining target recycling with Fe3O4/CeO2@Au nanocatalysts for microRNA-21 detection.

In this study, an electrochemical biosensor was proposed for microRNA-21 detection based on Fe3O4/CeO2 @Au magnetite nanoparticles (Fe3O4/CeO2 @Au MNPs) as nanocatalyst and catalytic hairpin assembly (CHA) for signal application. Firstly, target microRNA-21 hybridized with hairpin H2 to form H2-T duplex stranded DNA (dsDNA), which could further open the hairpin H1 for the formation of H1-H2 dsDNA. Simultaneously, the Fe3O4/CeO2 @Au-S1 not only hybridized with single stranded fragment of H1-H2 dsDNA with producing long dsDNA to absorb a large amount of electroactive substances of methylene blue (MB), but also acted as nanocatalyst to directly catalyze the reduction of MB for amplifying the electrochemical signal. Herein, compared with pure Fe3O4 nanoparticles, Fe3O4/CeO2 @Au MNPs exhibited excellent catalytic performance since the cerium oxide (CeO2) nanoparticles and Au nanoparticles can greatly improve the catalytic activity of Fe3O4 nanoparticles and effectively prevent the agglomeration of Fe3O4 nanoparticles. Owing to the signal amplification strategy, the proposed biosensor provided a wide linear range of 1 fM to 1 nM with a low detection limit of 0.33 fM (defined as S/N = 3) for microRNA-21 detection, and exhibited excellent specificity and sensitivity. This strategy provided a novel avenue for the detection of other biomarkers in electrochemical biosensors.