Ferrocene-labeled DNA Research Articles

Thermodynamic-assisted highly effective target converting strategy for sensitive electrochemical detection of trace lead (II) ion

In the present work, a thermodynamic-assisted target-converting strategy was designed by utilizing new 2D DNA nanoprobes (DNPs) to develop electrochemical biosensors for the sensitive determination of trace Pb2+. Herein, a stable DNP with a special structure was prepared through the hybridization reaction between three single-strand DNA (RS1, RS2, and P). Once the existence of Pb2+, the DNP will crumble and then release RS1 and RS2 due to the strong thermodynamic instability of the RS1/RS2 duplex. since RS1 and RS2 were designed in the same sequence in the free domain, they can be simultaneously used as effective output DNA, enhancing the conversion rate of Pb2+to ssDNA up to 1:2. Following the same principle, ferrocene-labeled bipedal DNPs (Fc-bDNPs) are prepared for stable electrochemical signal output. In the presence of RS1, RS2, and a hairpin DNA strand (H1), the ferrocene-labeled DNA strands (S3) escape from the Fc-bDNPs due to strand-displacement reaction, accompanying release of the two ssDNA outputs that can simultaneously trigger another strand-displacement reaction. Consequently, the one Pb2+input makes multiple Fc-labeled DNA strands far away from the electrode surface, resulting in a dramatic decrease in the response current of Fc. The developed biosensors show a linear relationship between current value and logarithmic concentration of Pb2+ ranging from 1 pM to 40 nM with a detection limit of 0.30 pM.

Electrochemiluminescence detection of miRNA-21 based on dual signal amplification strategies: Duplex-specific nuclease -mediated target recycle and nicking endonuclease-driven 3D DNA nanomachine

DNA nanomachines have shown potential application in the construction of various biosensors. Here, an electrochemiluminescence biosensor for the sensitive detection of miRNA-21 were reported based on three-dimensional (3D) DNA nanomachine and duplex-specific nuclease (DSN)-mediated target recycle amplification strategy. First, the bipedal DNA walkers were obtained by DSN-mediated digestion reaction initiated by target miRNA-21.3D DNA tracks were prepared by modifying Fe3O4 magnetic beads (MBs) with ferrocene-labeled DNA (Fc-DNA). The produced DNA walkers autonomously moved along 3D DNA tracks powered by nicking endonuclease. During the movement, ferrocene-labeled DNA was cleaved, resulting in large amounts of Fc-labeled DNA fragments away from the MBs surface. Finally, the liberated Fc-labeled DNA fragments were dropped on the C-g-C3N4 modified electrode surface, leading to the quenching of C-g-C3N4 electrochemiluminescence (ECL). Benefiting from the dual amplification strategy of 3D DNA nanomachine and DSN-mediated target recycling, the developed ECL biosensor exhibited an excellent performance for miRNA-21 detection with a wide linear range of 10 fM to 10 nM and a low detection limit of 1.0 fM. This work offers a new thought for the application of DNA walkers in the construction of various biosensors.

A Sensitive Signal-on Supersandwich DNA Biosensor Based on the Enhancement of Poly(aniline-luminol) Nanowires Electrochemiluminescence by Ferrocene.

A signal-on supersandwich type of electrochemiluminescence (ECL) DNA biosensor was developed based on the poly(aniline-luminol) nanowires (PALNWs) modified electrode and enhancement of ferrocene (Fc) on ECL of luminol. Aminated capture DNA was covalently linked to the PALNWs on the electrode surface by the crosslinking of glutaraldehyde. In presence of target DNA, its 3' terminus hybridizes with the capture probe and the 5' terminus hybridizes with ferrocene labeled DNA (Fc-DNA) to form a long DNA concatamer supersandwich structure. The ECL intensity of the prepared biosensor was clearly improved by increasing the concentration of target DNA due to the enhancement of ferrocene on luminol ECL. The difference of the ECL intensity in the absence and presence of target DNA was used to monitor the hybridization event. The difference of ECL linearly increased with the logarithm of target DNA concentration in the range from 1.0 × 10-16 - 1.0 × 10-8 mol L-1 with a detection limit of 5.8 × 10-17 mol L-1. The sensor had high sensitivity and wide linear relationship for the detection of target DNA.

Telomerase-triggered DNAzyme spiders for exponential amplified assay of cancer cells

A highly flexible electrochemical assay based on target-triggered DNAzyme spiders was proposed for the detection of telomerase. The DNAzyme-telomerase substrate primers (D-TSP) containing Cu2+-dependent DNAzymes serve as recognition elements, and primers of telomerase. Telomerase extracted from Hela cells recognize the D-TSP and elongated with DNA sequence repeats. A synthetic telomerase product hybridized with scaffold sequences of two DNAzyme-tethered probes on the basis of the mechanism of the proximity-ligation assay. The three-leg DNAzyme spiders has been assembled and initiated the autonomous hybridization/nicking/displacement cycles on substrate modified surface. The cleaved ferrocene-labeled fragements are adsorbed on gold surface leading to an increase in the electrochemical signal. As a result, the one input target, telomerase, release large amount of ferrocene-labeled DNA strands, achieving an exponential signal amplification and an excellent improvement in sensitivity over single molecule or two-component ‘sandwich’ binding complexes. Our proposed biosensor showed a nonlinear dependence with Hela cell numbers, ranging from 25 to 2000 with a detection limit of 10 cells. Telomerase activities from different cell lines were also successfully evaluated. Our electrochemical strategy based on target-triggered DNAzyme spiders was enzyme-free, PCR-free, simple in operation which indicated that it expected to expand the scope of DNA nanotechnology in the areas of clinical diagnosis.

Reversible and Distance-Controllable DNA Scissor: A Regenerated Electrochemiluminescence Biosensing Platform for Ultrasensitive Detection of MicroRNA.

Reversing the switching of DNA scissors with precisely control remains a compelling goal. Herein, based on strand displacement reaction within single step, the DNA scissor realized reversible switching and further controlled the distance of end strands along the movement of DNA scissor, which has been applied for the development of a regenerated sensing platform for the ultrasensitive detection of microRNA-21 (miRNA-21) with the electrochemiluminescence (ECL) complex (PEI-Ru(II)) as luminophores and diethylenetriamine (DETA) as the coreactant. In the presence of ferrocene-labeled DNA (Fc-DNA), the DETA-labeled DNA scissor clockwise switched to "off" state based on strand displacement reaction, resulting in the significant ECL quenching of Ru(II) system. Next, by using miRNA-21 as the motive fuel, the configuration of DNA scissor could be anticlockwise switched, which significantly enhanced the ECL intensity of Ru(II) complex due to the releasing of Fc-DNA and the proximity between DETA and Ru(II) complex. The reversible switching of DNA scissor led to the remarkably enhancing of ECL signal, realizing ultrasensitive detection of miRNA-21 with an excellent detection limit of 0.17 fM, which was also applied in miRNA detection successfully from different cancer cells. Impressively, the reversible switching of DNA scissor biosensor was able to realize the regeneration of the biosensing platform by adding an additional single stranded DNA (ssDNA) based on strand displacement reaction within a single step, providing a novel concept for constructing simple and sensitive regenerated biosensor.

Novel 2D-DNA-Nanoprobe-Mediated Enzyme-Free-Target-Recycling Amplification for the Ultrasensitive Electrochemical Detection of MicroRNA.

In this work, on the basis of a new 2D DNA nanoprobe (DNP) and an enzyme-free-target-recycling amplification, an electrochemical biosensor is developed for the ultrasensitive detection of microRNA-21 (miRNA-21). Herein, two ferrocene-labeled bipedal DNPs, which show small steric hindrance and strong stability, are prepared on the basis of the mechanism of the proximity-ligation assay (PLA), improving the space utilization. In the presence of the target, miRNA-21, and a hairpin DNA strand, the DNP will collapse, and then two ferrocene-labeled DNA strands and the miRNA-21 will be simultaneously released from the electrode surface through toehold-mediated strand-displacement reactions (TSDRs), leading to a decrease in the electrochemical signal and realization of enzyme-free target recycling. As a result, the one input target, miRNA-21, could release 2 N ferrocene-labeled DNA strands, achieving a dramatic decrease in the electrochemical signal. Combining DNPs and enzyme-free target recycling, this proposed biosensor showed a linear dependence with miRNA-21 concentration, ranging from 1.0 fM to 10 nM with a detection limit of 0.31 fM. In addition, it is worth mentioning that this biosensor can be regenerated through incubating with three assistant-DNA strands, realizing the reuse of raw materials. Surprisingly, the elaborated biosensor provides a novel strategy for building controllable DNA nanoprobes for the sensitive detection of various biomarkers.

An electrochemiluminescence biosensing platform for Hg2+ determination based on host–guest interactions between β-cyclodextrin functionalized Pd nanoparticles and ferrocene

A solid-state electrochemiluminescence (ECL) switch biosensor using a Ru(bpy)32+/β-cyclodextrin–Pd nanoparticles (β-CY–PdNPs)/gelatin (Gel) complex and a ferrocene-labeled DNA probe (Fer-DNA) for the detection of Hg2+ was successfully developed.

Ultrasensitive electrochemical biosensor for silver ion based on magnetic nanoparticles labeling with hybridization chain reaction amplification strategy

Silver ion (Ag+) is a highly toxic heavy metal ion to aquatic organisms and accumulates in the human body via the food chain. Therefore, fast and accurate detection of Ag+ in water and food resources has become a critical issue within the scope of human health. Herein, we developed an ultrasensitive electrochemical biosensor for detection of Ag+ based on magnetic Fe3O4@gold core-shell nanoparticles (Fe3O4@Au NPs) labeling with hybridization chain reaction (HCR) amplification strategy. In this sensing strategy, the magnetic Fe3O4@Au NPs were selected for labeling with HCR product and enrichment on the surface of magnetic gold electrode. Thiolated-oligonucleotide (S1) was firstly immobilized on the surface of Fe3O4@Au NPs through Au–S chemical bond. In the presence of Ag+, cytosine-rich DNA oligonucleotide S2 hybridized with S1 to form an intramolecular duplex, in which Ag+ can selectively bind to cytosine–cytosine mismatches forming C–Ag+–C complex. The exposed stem of the C–Ag+–C complex opened two alternating ferrocene-labeled DNA hairpins (H1 and H2) in turn and triggered HCR to form a supersandwich DNA structure on the surface of Fe3O4@Au NPs. The HCR products modified Fe3O4@Au NPs were brought to the surface of magnetic gold electrode for direct electrochemical measurements. The proposed strategy led to a low detection limit of 0.5fM and a wide dynamic range of 1fM–100pM for target Ag+. The developed biosensor was highly selective and its practical applicability in tap water and lake water samples was also investigated with a satisfactory result.

Signal enhancement in ultraflat electrochemical DNA biosensors.

The ability of holding back the undesired molecules, but at the same time to provide the right distribution and orientation of the bioreceptors, are critical targets to reach an efficient hybridization and enhanced detection in electrochemical DNA biosensors. The main actors responsible of these key functions are the substrate of the sensor and the interface auto-assembled on it. In this paper we present the annealing as a method to improve commercial gold evaporated substrates for biosensor applications. The restructuring of granulated gold surface by means of annealing heating treatment leads to the formation of ultraflat gold lamellar terraces. The formation of terraces was characterized with scanning tunneling microscopy and optical interferometry. The performance of the sensor sensitivity on granular substrates and ultraflat substrates was studied, concerning the orientation and surface coverage of the bioreceptor interface applied in electrochemical biosensor. The hybridization efficiency of ferrocene-labeled DNA amplified by PCR was characterized with surface plasmon resonance and electrochemistry. The experimental results demonstrate that annealing process, positive influence on optical and voltammetric readings, due to a structured organization of the bioreceptors on the flat substrate, gaining more efficient immobilization and DNA hybridization. The results suggest the annealing as a powerful tool for improving gold substrates in biosensors applications.

Effect of Ferrocene Position on Charge Transfer in ds-DNA Films

Ferrocene-labeled DNA systems where the Fc group is directly attached to deoxyuridine are investigated electrochemically in an effort to elucidate the electron transfer properties of these systems. The rigid linkage between the Fc group and the nucleobase provides control over the position of the Fc group within the ds-DNA and helps to elucidate the effect of the position of the redox label on the electrochemical behaviour of the ds-DNA films. Cyclic voltammetry, electrochemical surface plasmon resonance and scanning electrochemical microscopy measurements show that charge transfer (CT) in such systems is to a large extent controlled by the dynamic properties of surface-bound ds-DNA rather than by the CT through base-pair stacks.

A regenerative electrochemical sensor based on oligonucleotide for the selective determination of mercury(ii)

We have developed a selective, sensitive, and re-usable electrochemical sensor for Hg2+ ion detection. This sensor is based on the Hg2+-induced conformational change of a single-stranded DNA (ssDNA) which involves an electroactive, ferrocene-labeled DNA hairpin structure and provides strategically the selective binding of a thymine-thymine mismatch for the Hg2+ ion. The ferrocene-labeled DNA is self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface blocked with 3-mercapto-1-propanol to form a mixed monolayer. The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety. The 'signal-on' upon mercury binding could be attributed to a change in the conformation of ferrocene-labeled DNA from an open structure to a restricted hairpin structure. The differential pulse voltammetry (DPV) of the modified electrode showed a linear response of the ferrocene oxidation signal with increase of Hg2+ concentration in the range between 0.1 and 2 microM with a detection limit of 0.1 microM. The molecular beacon mercury(II) ion sensor was amenable to regeneration by simply unfolding the ferrocene-labeled DNA in 10 microM cysteine, and could be regenerated with no loss in signal gain upon subsequent mercury(II) ion binding.

A Controllable Solid-State Ru(bpy)32+ Electrochemiluminescence Film Based on Conformation Change of Ferrocene-Labeled DNA Molecular Beacon

A controllable solid-state electrochemiluminescence (ECL) film based on efficient and stable quenching of ECL of ruthenium(II) tris-(bipyridine) (Ru(bpy)32+) by oxidizing ferrocene (Fc) at the electrode is developed. The ECL intensity is correlated to the distance which is controlled by the conformation of the ferrocene-labeled DNA molecular beacon (Fc-MB) between the Fc and Ru(bpy)32+ immobilized on the electrode. The conformation adjustment is conducted via complementary DNA hybridizing with the bases in the loop of the Fc-MB and changing the temperature of the Fc-MB and the resultant double-stranded DNA (dsDNA). Those events all result in change of the ECL intensity. With such characteristics, the solid-state Ru(bpy)32+-ECL film has the potential to be applied to reagentless DNA ECL biosensors and to calculate thermodynamic parameters of equilibrium constants of MB binding and the stem-loop formation.