A Peptide Cleavage-Based Ultrasensitive Electrochemical Biosensor with an Ingenious Two-Stage DNA Template for Highly Efficient DNA Exponential Amplification

The direct transduction of a peptide cleavage event into DNA detection has always produced output DNA with some amino acid residues, which influence the DNA amplification efficiency in view of their steric hindrance effect. Here an ingenious two-stage DNA template was designed to achieve highly efficient DNA amplification by utilizing the DNA exponential amplification reaction (EXPAR) as a model. The usage of a two-stage DNA template not only accomplished the traditionally inefficient EXPAR triggered by output DNA with some amino acid residues but also simultaneously produced a newly identical DNA trigger without any amino acid residues to induce an extra efficient EXPAR, which significantly improved the DNA amplification efficiency, realizing the ultrasensitive detection of the target. On the basis of the proposed highly efficient DNA amplification strategy, a novel peptide cleavage-based electrochemical biosensor was constructed to ultrasensitively detect matrix metalloproteinases-7 (MMP-7). As a result, this developed assay demonstrated excellent sensitivity with a linear range from 0.1 pg·mL^–1 to 50 ng·mL^–1 and a detection limit down to 0.02 pg·mL^–1, which paved a novel avenue for constructing ultrasensitive peptide cleavage-based biosensors

Highly Efficient Target Recycling-Based Netlike Y‑DNA for Regulation of Electrocatalysis toward Methylene Blue for Sensitive DNA Detection

In this work, the highly efficient target recycling-based netlike Y-shaped DNA (Y-DNA), which regulated the electrocatalysis of Fe₃O₄@CeO₂–Pt nanoparticles (Fe₃O₄@CeO₂–PtNPs) toward methylene blue (MB) for signal amplification, was developed to prepare a sensitive DNA biosensor for detecting the DNA associated with oral cancer. Specifically, with the help of highly efficient enzyme-assisted target recycling (EATR) amplification strategy, one target DNA input was converted to corresponding plenty of DNA strands S1Fe₃O₄@CeO₂–Pt and S2MB output, which could be employed to interact with HP2 immobilized on the electrode surface to form stable netlike Y-DNA without any waste of recycling products. Meanwhile, the formation of netlike Y-DNA could regulate electrocatalytic efficiency of Fe₃O₄@CeO₂–PtNPs, inducing the proximity of Fe₃O₄@CeO₂–PtNPs to MB and significantly enhancing electrochemical signal. Further, the signal could also be amplified by Fe₃O₄@CeO₂–PtNPs modified on the electrode surface. By virtue of this ingenious design, a novel netlike Y-DNA structure based on highly efficient EATR was simply constructed and successfully applied to an electrochemical DNA biosensor along with electrocatalysis of Fe₃O₄@CeO₂–PtNPs, achieving the sensitive detection of target DNA ranging from 10 fM to 50 nM with a detection limit of 3.5 fM. Impressively, the biosensor here demonstrates an admirable method for regulating the electrocatalysis of NPs toward substrates to enhance signal, and we believe that this biosensor is a potential candidate for the sensitive detection of target DNA or other disease-related nucleic acids

Bi-directional DNA Walking Machine and Its Application in an Enzyme-Free Electrochemiluminescence Biosensor for Sensitive Detection of MicroRNAs

Herein, a dual microRNA (miRNA) powered bi-directional DNA walking machine with precise control was developed to fabricate an enzyme-free biosensor on the basis of distance-based electrochemiluminescence (ECL) energy transfer for multiple detection of miRNAs. By using miRNA-21 as the driving force, the DNA walker could move forth along the track and generated quenching of ECL response due to the proximity between Au nanoparticles (AuNPs) and Mn²⁺ doped CdS nanocrystals (CdS:Mn NCs) film as the ECL emitters, realizing ultrasensitive determination of miRNA-21. Impressively, once miRNA-155 was introduced as the driving force, the walker could move back along the track automatically, and surface plasmon resonance (SPR) occurred owing to the appropriate large separation between AuNPs and CdS:Mn NCs, achieving an ECL enhancement and realizing ultrasensitive detection of miRNA-155. The bi-directional movement of the DNA walker on the track led to continuous distance-based energy transfer from CdS:Mn NCs film by AuNPs, which resulted in significant ECL signal variation of CdS:Mn NCs for multiple detection of miRNA-21 and miRNA-155 down to 1.51 fM and 1.67 fM, respectively. Amazingly, the elaborated biosensor provided a new chance for constructing controllable molecular nanomachines in biosensing, disease diagnosis, and clinical analysis

Biodegradable MnO₂ Nanosheet-Mediated Signal Amplification in Living Cells Enables Sensitive Detection of Down-Regulated Intracellular MicroRNA

The monitoring of intracellular microRNAs plays important roles in elucidating the biological function and biogenesis of miRNAs in living cells. However, because of their sequence similarity, low abundance, and small size, it is a great challenge to detect intracellular miRNAs, especially for those with much lower expression levels. To address this issue, we have developed an in cell signal amplification approach for monitoring down-regulated miRNAs in living cells based on biodegradable MnO₂ nanosheet-mediated and target-triggered assembly of hairpins. The MnO₂ nanosheets can adsorb and exhibit an excellent quenching effect to the dye labeled hairpin probes. Besides, due to their biodegradability, the MnO₂ nanosheets feature highly reduced cytotoxicity to the target cells. Upon entering cells, the surface-adsorbed FAM- and Tamra (TMR)-conjugated hairpins can be released due to the displacement reactions by other proteins or nucleic acids and the degradation of the MnO₂ nanosheets by cellular GSH. Subsequently, the down-regulated target miRNA-21 triggers cascaded assembly of the two hairpins into long dsDNA polymers, which brings the fluorescence resonance energy transfer (FRET) pair, FAM (donor), and TMR (acceptor) into close proximity to generate significantly enhanced FRET signals for detecting trace miRNA-21 in living cells. By carefully tailoring the sequences of the hairpins, the developed method can offer new opportunities for monitoring various trace intracellular miRNA targets with low expression levels in living cells

An “Off–On” Electrochemiluminescent Biosensor Based on DNAzyme-Assisted Target Recycling and Rolling Circle Amplifications for Ultrasensitive Detection of microRNA

In this study, an off–on switching of a dual amplified electrochemiluminescence (ECL) biosensor based on Pb²⁺-induced DNAzyme-assisted target recycling and rolling circle amplification (RCA) was constructed for microRNA (miRNA) detection. First, the primer probe with assistant probe and miRNA formed Y junction which was cleaved with the addition of Pb²⁺ to release miRNA. Subsequently, the released miRNA could initiate the next recycling process, leading to the generation of numerous intermediate DNA sequences (S2). Afterward, bare glassy carbon electrode (GCE) was immersed into HAuCl₄ solution to electrodeposit a Au nanoparticle layer (depAu), followed by the assembly of a hairpin probe (HP). Then, dopamine (DA)-modified DNA sequence (S1) was employed to hybridize with HP, which switching off the sensing system. This is the first work that employs DA to quench luminol ECL signal, possessing the biosensor ultralow background signal. Afterward, S2 produced by the target recycling process was loaded onto the prepared electrode to displace S1 and served as an initiator for RCA. With rational design, numerous repeated DNA sequences coupling with hemin to form hemin/G-quadruplex were generated, which could exhibit strongly catalytic toward H₂O₂, thus amplified the ECL signal and switched the ON state of the sensing system. The liner range for miRNA detection was from 1.0 fM to 100 pM with a low detection limit down to 0.3 fM. Moreover, with the high sensitivity and specificity induced by the dual signal amplification, the proposed miRNA biosensor holds great potential for analysis of other interesting tumor markers

An Electrochemical Biosensor for Sensitive Detection of MicroRNA-155: Combining Target Recycling with Cascade Catalysis for Signal Amplification

In this work, a new electrochemical biosensor based on catalyzed hairpin assembly target recycling and cascade electrocatalysis (cytochrome <i>c</i> (Cyt <i>c</i>) and alcohol oxidase (AOx)) for signal amplification was constructed for highly sensitive detection of microRNA (miRNA). It is worth pointing out that target recycling was achieved only based on strand displacement process without the help of nuclease. Moreover, porous TiO₂ nanosphere was synthesized, which could offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA strand 1 (S1) and Cyt <i>c</i> accordingly. With the mimicking sandwich-type reaction, the cascade catalysis amplification strategy was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H₂O₂, which was further electrocatalyzed by PtNPs and Cyt <i>c</i>. This newly designed biosensor provided a sensitive detection of miRNA-155 from 0.8 fM to 1 nM with a relatively low detection limit of 0.35 fM

Host–Guest Recognition-Assisted Electrochemical Release: Its Reusable Sensing Application Based on DNA Cross Configuration-Fueled Target Cycling and Strand Displacement Reaction Amplification

In this work, an elegantly designed host–guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p (miRNA-182-5p), a prostate cancer biomarker in prostate cancer, based on the DNA cross configuration-fueled target cycling and strand displacement reaction (SDR) amplification. With such a design, the single target miRNA input could be converted to large numbers of single-stranded DNA (S1-Trp and S2-Trp) output, which could be trapped by cucurbit[8]­uril methyl viologen (CB-8-MV²⁺) based on the host–guest recognition, significantly enhancing the sensitivity for miRNA detection. Moreover, the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently, avoiding the waste and saving the experiment cost. Impressively, by resetting a settled voltage, the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface, attributing that the guest ion methyl viologen (MV²⁺) was reduced to MV^+<b>·</b> under this settled voltage and formed a more-stable CB-8-MV^+<b>·</b>–MV^+<b>·</b> complex. Once O₂ was introduced in this system, MV^+<b>·</b> could be oxidized to MV²⁺, generating the complex of CB-8-MV²⁺ for capturing S1-Trp and S2-Trp again in only 5 min. As a result, the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release, overcoming the challenges of time-consuming, burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor. Furthermore, the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%–102.24%. After all, the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of nucleic acids or proteins

High-Sensitive Electrochemiluminescence C‑Peptide Biosensor via the Double Quenching of Dopamine to the Novel Ru(II)-Organic Complex with Dual Intramolecular Self-Catalysis

Here, a novel Ru­(II)-organic complex (Ru-PEI-ABEI) with high electrochemiluminescence (ECL) efficiency was proposed to construct a sensitive quenching-typed ECL biosensor for C-peptide (C–P) measurement based on the double quenching effect of dopamine (DA). The high ECL efficiency of Ru-PEI-ABEI was originated from the dual intramolecular self-catalysis including intramolecular coreaction between polyethylenimine (PEI) and Ru­(bpy)₂(mcbpy)²⁺, and intramolecular ECL resonance energy transfer (ECL-RET) from <i>N</i>-(aminobutyl)-<i>N</i>-(ethylisoluminol) (ABEI) to Ru­(bpy)₂(mcbpy)²⁺, which would generate a strong initial ECL signal. Through sandwiched immunoreaction and 3D DNA walking machine, a certain amount of target C–P was converted to a large amount of intermediate DNA that could further trigger hybridization chain reaction (HCR) to introduce into massive DA which not only could quench the ECL of Ru­(bpy)₂(mcbpy)²⁺, but also quench the ECL of ABEI. Thus, the double quenching effect of DA would effectively quench the ECL of Ru-PEI-ABEI, leading to an obviously decreased final ECL signal. Thus, a sensitive quenching-typed ECL biosensor was constructed for C–P detection with a linear range from 50 fg mL^–1 to 16 ng mL^–1 and an estimated detection limit of 16.7 fg mL^–1. The dual intramolecular self-catalyzed strategy and the double quenching strategy based on one quencher to the same luminous reagent proposed in this work would provide new thought in both ECL signal enhancement and quenching efficiency improvement

A DNA-Fueled and Catalytic Molecule Machine Lights Up Trace Under-Expressed MicroRNAs in Living Cells

The detection of specific intracellular microRNAs (miRNAs) in living cells can potentially provide insight into the causal mechanism of cancer metastasis and invasion. However, because of the characteristic nature of miRNAs in terms of small sizes, low abundance, and similarity among family members, it is a great challenge to monitor miRNAs in living cells, especially those with much lower expression levels. In this work, we describe the establishment of a DNA-fueled and catalytic molecule machinery in cell signal amplification approach for monitoring trace and under-expressed miRNAs in living cells. The presence of the target miRNA releases the hairpin sequences from the dsDNA (containing the fluorescence resonance energy transfer (FRET) pair-labeled and unfolded hairpin sequences)-conjugated gold nanoparticles (dsDNA-AuNPs), and the DNA fuel strands assist the recycling of the target miRNA sequences via two cascaded strand displacement reactions, leading to the operation of the molecular machine in a catalytic fashion and the release of many hairpin sequences. As a result, the liberated hairpin sequences restore the folded hairpin structures and bring the FRET pair into close proximity to generate significantly amplified signals for detecting trace miRNA targets. Besides, the dsDNA-AuNP nanoprobes have good nuclease stability and show low cytotoxicity to cells, and the application of such a molecular system for monitoring trace and under-expressed miRNAs in living cells has also been demonstrated. With the advantages of in cell signal amplification and reduced background noise, the developed method thus offers new opportunities for detecting various trace intracellular miRNA species

Sensitive Electrochemiluminescence Immunosensor for Detection of <i>N</i>‑Acetyl-β‑d‑glucosaminidase Based on a “Light-Switch” Molecule Combined with DNA Dendrimer

Here, a novel “light-switch” molecule of Ru (II) complex ([Ru­(dcbpy)₂dppz]²⁺-DPEA) with self-enhanced electrochemiluminescence (ECL) property is proposed, which is almost nonemissive in aqueous solution but is brightly luminescent when it intercalates into DNA duplex. Owing to less energy loss and shorter electron-transfer distance, the intramolecular ECL reaction between the luminescent [Ru­(dcbpy)₂dppz]²⁺ and coreactive tertiary amine group in <i>N</i>,<i>N</i>-diisopropylethylenediamine (DPEA) makes the obtained “light-switch” molecule possess much higher light-switch efficiency compared with the traditional “light-switch” molecule. For increasing the loading amount and further enhancing the luminous efficiency of the “light-switch” molecule, biotin labeled DNA dendrimer (the fourth generation, G₄) is prepared from Y-shape DNA by a step-by-step assembly strategy, which provides abundant intercalated sites for [Ru­(dcbpy)₂dppz]²⁺-DPEA. Meanwhile, the obtained nanocomposite (G₄-[Ru­(dcbpy)₂dppz]²⁺-DPEA) could well bind with streptavidin labeled detection antibody (SA-Ab2) due to the existence of abundant biotin. Through sandwiched immunoreaction, an ECL immunosensor was fabricated for sensitive determination of <i>N</i>-acetyl-β-d-glucosaminidase (NAG), a typical biomarker for diabetic nephropathy (DN). The detemination linear range was 0.1 pg mL^–1 to 1 ng mL^–1, and the detection limit was 0.028 pg mL^–1. The developed strategy combining the ECL self-enhanced “light-switch” molecular and DNA nanotechnology offers an effective signal amplification mean and provides ample potential for further bioanalysis and clinical study