Search

In this work, a dual amplified signal enhancement approach based on coupling deoxyribozyme (DNAzyme)-driven bipedal DNA walkers (BDW) and terminal deoxynucleotidyl transferase (TdT)-mediated DNA elongation signal amplifications has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. In presence of thrombin, the BDW complex, which is comprised from the target thrombin and two DNAzyme-containing probes, can exhibit autonomous cleavage behavior on the surface of the substrate DNA (SD) modified electrode, and remove the cleaved DNA fragment from the electrode surface. Subsequently, the TdT can catalyze the elongation of the SD with free 3'-OH termini and formation of many G-quadruplex sequence replicates with the presence of 2'-deoxyaguanosine-5'-triphosphate (dGTP) and adenosine 5'-triphosphate (dATP) at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a "signal-on" and completely label-free fashion. Under optimized conditions, the response peak current was linear with the concentration of thrombin in the range from 0.5 pM to 100000 pM with detection limit of 0.31 pM. This research provides us a sustainable idea for the hyphenated multiple amplification strategies and a stable and effective method for the detection of protein biomarkers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein, a robust and highly ordered three-dimensional electrochemical DNA (3D E-DNA) biosensor was proposed, and its orientation was controlled from top down by poly adenine oligonucleotides (polyA-ODNs)-mediated rolling motor (PRM) and graftable tetrahedron DNA (GTD). The GTD with a grafting domain was immobilized on the electrode surface to construct a well-organized sensing interface and controlled the orientation and distribution of the whole system at the "bottom" of this biosensor. The polyA-ODNs regulated the direction and density of the leg DNA attached on PRM at the "top" of the biosensor. The motion was achieved through the target induced cyclic cleaving, which triggered the motor rolling rather than walk. Impressively, the duplex strand DNA (dsDNA) formed after grafting, as a girder, provided a stable support to the soft long single strand (ssDNA), which facilitated the formation of the catalytic center, elevated the efficiency of the rolling cleavage. Under the optimal conditions, the designed biosensor exhibited a lower limit of 0.17 nM and wide linear range from 0.5 nM to 1.5 μM for adenosine rapid detection. Unique dual orientation regulated characteristics of the system increased the probability hybridization enormously and improved the motion efficiency significantly, which offered new avenue of DNA nanomachines development in biosensor platform., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein, a signal-on electrochemical aptasensor for highly sensitive detection of thrombin (TB) was constructed based on the DNAzyme-driven DNA walker strategy. We developed a new dual functional hairpin DNA (HP) containing a substrate sequence of the Mg 2+ -dependent DNAzyme (in the loop region) and the G-quadruplex forming segment (in the stem region). The DNA walker (TBA 2 -DWs), containing a TB aptamer and an enzymatic sequence, was introduced onto gold electrode (GE) by aptamers-target specific recognition, and thus initiated the enzymatic sequences to hybridize with the substrate sequence. Then, the DNA walker could repeatedly bind and cleave HP in the assistance of Mg 2+ , unlocking many active G-quadruplex forming sequences. Finally, hemin can further bind the G-quadruplex to form G-quadruplex/hemin complexes and generate enhanced current output. The aptasensor for TB assay showed a linear detection range from 1 pM to 60000 pM with a lower detection limit of 0.58 pM. And more, the proposed detection strategy was enzyme-free and label-free., (Copyright © 2019 Elsevier B.V. All rights reserved.)

RecA of Escherichia coli and its active nucleoprotein filaments with DNA are important for the genomic integrity and the genetic diversity. The formation of the DNA-RecA nucleoprotein filaments is a complex multiple-step process and can be affected by many factors. In this work, the effects of poly-L-lysine (PLL) on the DNA-RecA nucleoprotein filaments are investigated in vitro by agarose gel electrophoresis and atomic force microscopy (AFM). The observed morphologies vary with the concentration, the length, and the addition order of PLL. These distinctions provide information for the conformation change of DNA and the binding sites of RecA protein in the formation process of nucleoprotein filaments. Besides that, the comparison displays the effect of cationic polyelectrolyte on both inhibition and destabilization of the DNA-RecA nucleoprotein filaments. The current study provides valuable insights for pharmacologic studies and gene delivery.

In this work, we illustrate a simple chelation-based strategy to trigger DNA release from DNA-incorporated multilayer films, which were fabricated through the layer-by-layer (LbL) assembly of DNA and inorganic zirconium (IV) ion (Zr(4+)). After being incubated in several kinds of chelator solutions, the DNA multilayer films disassembled and released the incorporated DNA. This was most probably due to the cleavage of coordination/electrostatic interactions between Zr(4+) and phosphate groups of DNA. Surface plasmon resonance (SPR), UV-vis spectrometry and atomic force microscopy (AFM) were used to characterize the assembly and the disassembly of the films. By incorporating plasmid DNA (pDNA) into this controllable disassembly system, the multilayer films sustained the consecutive DNA release. The released pDNA retained its integrity and transcriptional activity, and also expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. Besides the simplicity and cost efficiency of this method, the most advantage of this route was that the release of DNA from the films could be modulated by various external conditions, such as the chelator and ionic strength. The Zr(4+)/DNA multilayer films with the ability to precisely control the release rate of DNA might serve as an alternative localized gene delivery system in the perspective of biomedical applications.

A layer-by-layer film composed of DNA and inorganic zirconium ion (Zr(4+)) was fabricated on the surface of gold thin film, and an electric field triggered disintegration of the multilayer film was studied by using electrochemical surface plasmon resonance (EC-SPR). EC-SPR results demonstrated that the film was disassembled upon the application of an electric field and the disassembly rate varied with the applied potential, leading to the controlled release of DNA. The electrodissolution could be switched off by removing the electric potential and reactivated by reapplying the potential. By incorporating plasmid DNA (pDNA) in to this controlled release system, the multilayer film could sustain the consecutive release of pDNA electrochemically. The released pDNA retained its integrity and transfection activity, and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The electrochemical systems, with advantages of miniaturization, surface-tailoring, safety, simplicity, convenience, automation, low-cost, and free of immune reactions, made the electrical route a very attractive gene-delivery alternative.

DNA was efficiently bound to water-soluble positively charged CdTe quantum dots (QDs) through complementary electrostatic interaction. These QDs-DNA complexes were disrupted and DNA was released by glutathione (GSH) at intracellular concentrations. Interestingly, there was almost no detectable DNA released by extracellular concentration of GSH. The formation of QDs-DNA complexes and GSH-mediated DNA release from the complexes were confirmed by dye displacement assay, electrophoretic mobility shift assay (EMSA), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) experiments. The released DNA retained transcriptional activity and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The transfection efficiency measured by flow cytometry (FCM) was comparable with the positive control. The obvious difference of GSH concentration in nature between the intra- and extracellular environments as well as the GSH concentration-dependent triggered release suggests potential applications of these positively QDs in selective unpacking of payload in living cells in a visible manner.

• Triplex DNA molecular switch recognize different targets and release the same probe molecular. • CuInS 2 /rGO was used to enhance the photocurrent signal. • A universal photoelectrochemical aptasensor was constructed for antibiotic highly selective and sensitive detection. In this work, a universal and convenient photoelectrochemical (PEC) biosensor was proposed to detect antibiotic on the basis of CuInS 2 /rGO and triplex DNA molecular switch. High performance photoactive structure was formed via p-type CuInS 2 nanosphere complexed with rGO, which was employed to enhance the cathode photocurrent. Capture probe (CP) was conjugated onto the photoactive interface through covalent bonding. In the absence of target, the triplex DNA (THMS) was in a closed state. After introducing a target, the interaction between the target and the specific ring part DNA fragment leaded to the dissociation of the THMS and thereby liberated the single strand DNA (SP). The above identification process was completed in the homogeneous solution. Then, SP was captured to the electrode by CP and hybridized to form dsDNA. The steric effect of dsDNA hindered the interfacial charge transfer, resulting in signal-off photocurrent output. By only altering the specific loop section sequence of THMS, different targets can release the same SP, and can be detected on the same sensing platform under the same testing conditions. As proof of principle, three kinds of antibiotics amoxicillin (AMOX), ampicillin (AMP) and tobramycin (TOB) was detected, achieving limit of detection values of 3.17 pM, 0.63 nM and 2.81 fM, respectively. This approach was also used to detect antibiotic residues in food and environmental samples, which provides a promising paradigm for developing universal, high-performance and convenient PEC biosensing platform. [ABSTRACT FROM AUTHOR]

• P-type CuInS 2 microflower recombined rGO modified ITO was used as photocathode. • Target was recognized by triple helix DNA switch in homogeneous solution. • Ferrocene labeled single stranded DNA was released as signal probe. • Energy level matching between ferrocene and CuInS 2 leaded an enhanced photocurrent. A sensitive signal-on photoelectrochemical aptasensor for antibiotic determination was constructed based on the energy level matching between ferrocene and CuInS 2. P-type CuInS 2 microflower was complexed with reduced graphene oxide (CuInS 2 /rGO) to get photocathode current with good photoelectric conversion efficiency and stability. Then, hairpin DNA (HP) was covalently bonded to the electrode surface. A triple helix DNA (THMS) was used as a molecular switch. After the specific recognition between target and THMS in homogeneous solution, ferrocene labeled probe (Fc-T2) was released. Finally, Fc-T2 was captured by the HP, which leaded the obvious increase of photocurrent for the energy level matching between ferrocene and CuInS 2. The increase of the photocurrent signal was proportional to the concentration of target amoxicillin (AMOX), the linear range was 100 fM-100 nM with detection limit of 19.57 fM. Meanwhile, the method has been successfully applied for milk and lake water samples analysis with satisfactory results. [ABSTRACT FROM AUTHOR]

In this work, a novel strategy for preparation of bipedal DNA walker (BDW) based on hybridization chain reaction (HCR) with the assistance of Exonuclease III (Exo III) was proposed. Based on this strategy, an electrochemical biosensor was constructed to achieve sensitive detection of CYFRA 21-1 DNA. Firstly, target recognition and circulation were achieved through a one-step catalytic hairpin assembly (CHA) reaction. For further amplification, hybridization chain reaction (HCR) was employed to form duplex-stranded DNA (dsDNA) nanostructure in homogeneous solution. In particular, the elongated single strand of the hairpin DNA for HCR was designed as the Mg2+ DNAzyme sequence. With the assistance of Exo III, dsDNA nanostructure can be digested and transformed into large amounts of BDW. These BDW can cleave the signal probe driven by Mg2+, which was modified on the electrode surface and thus achieved "signal-off" detection of target. This BDW preparation method based on HCR with the digestion of Exo III converted one target input into large amount of BDW. Coupled with the walking cleavage of BDW, a series of cascade amplification endowed high sensitivity with this biosensor and realized ultrasensitive detection of target DNA with the detection limit as low as 3.01 aM. [Display omitted] • The preparation of bipedal DNA walker (BDW) based on hybridization chain reaction (HCR) was firstly proposed. • One target input can be converted into large amount of BDW. • Triple signal amplification was achieved by integrating catalytic hairpin assembly (CHA), HCR and BDW. • This biosensor realized ultrasensitive detection of CYFRA 21-1 DNA with the detection limit as low as 3.01 aM. [ABSTRACT FROM AUTHOR]

Abstract: Here, we demonstrated dimethyldioctadecylammonium bromide (DODAB), a cationic lipid, bilayer coated Au nanoparticles (AuNPs) could efficiently deliver two types of plasmid DNA into human embryonic kidney cells (HEK 293) in the presence of serum. The transfection efficiency of AuNPs was about five times higher than that of DODAB. The interaction of AuNPs with DNA was characterized with dye intercalation assay and agarose gel electrophoresis. The morphology of the complex of AuNPs with DNA was observed with scanning electron microscope (SEM). The intracellular trafficking of the complex was monitored with transmission electron microscope (TEM). Based on experimental results, the possible mechanism was proposed and the barriers in the process of transfection were discussed. This work demonstrates a simple way to increase the transfection efficiency of cationic lipid through changing the stability of the complex of cationic lipid with DNA. It may provide some insights into understanding and controlling the interaction of cationic lipid with DNA. It also provides a novel way to construct gold nanoparticles-based gene vectors and some insights into learning the process of nanomaterials-mediated transfection. [Copyright &y& Elsevier]

A novel electrochemical biosensing strategy was proposed to detect cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA based on Exo III-assisted digestion of dsDNA polymer (EADDP) from hybridization chain reaction (HCR). Primarily, the presence of target can drive a catalytic hairpin assembly (CHA) reaction, which was aimed to achieve target recognition and circulation. Then the HCR can be triggered for further signal amplification and generate long dsDNA polymer with signal tags. Subsequently, the introduction of Exo III can digest the long dsDNA polymer to produce large amounts of double signal fragments (DSFs). The above experiments were all carried out in homogeneous solution. Finally, the released DSF can be captured onto the electrode directly by capture probe (CP) and a highly amplified electrochemical signal can be detected. The EADDP in homogeneous solution circumvented complex solid-liquid interface reaction and tedious operation steps on electrode. Besides, one target can be converted into abundant DSFs, which greatly improved the sensitivity. This biosensor exhibited a low detection limit (0.0348 fM) and wide linear range (5 fM ∼ 50 nM) for CYFRA 21-1 DNA biosensing with reliable specificity and stability. [Display omitted] • A novel strategy based on Exo III-assisted digestion of dsDNA polymer (EADDP) was proposed. • The Exo III was firstly used to digest dsDNA polymer to produce abundant double signal fragments (DSF). • The EADDP can convert one target into large amount of DSF in homogeneous solution. • EADDP in homogeneous solution and direct capture of DSF simplified the operation steps on electrode. • This biosensor achieved a low detection limit of 0.0348 fM for CYFRA 21-1 DNA detection. [ABSTRACT FROM AUTHOR]

• A novel structural of DNA nanotweezer is designed to construct electrochemical DNA biosensor. • Split G-rich sequences on the periphery of DNT is not only used as signal generator but also can reduce the background signal to improve sensitivity. • The preparation of this biosensor is convenient and inexpensive. • This DNA biosensor has high sensitivity, well stability and reproducibility for circulating tumor DNA detection. In this work, a label-free electrochemical biosensor for DNA assay was constructed based on a novel structural DNA nanotweezer (DNT) coupled with split G-quadruplex. The novel DNT consists of a long single strand with two hairpin structures in the middle as a recognition element and two short single strands with split G-rich sequences at outsides as a signal reporter. The target triggered two hairpin structures hybridized with each other, it caused the DNT transformation from "open" to "close". The "closed" DNT facilitated the split G-rich sequences combined together and provided electrochemical signals. Here, the hairpin structures were not only used for specific target identification but also avoided the false positive signals and improved the sensitivity. Additionally, the split G-rich sequences were put at outsides instead of insides, which significantly improved the signal-background ratio. Moreover, the construction of this biosensor was convenient and easy to control, which was beneficial to reduce the error generated by the operation process and improve stability and reproducibility. Under the optimal conditions, the proposed assay achieved a low detection limit (22 aM) and a wide linear range (1 fM to 1 nM) for circulating tumor DNA determination. This efficient and convenient DNT can be used as a template in electrochemical biosensors fabrication. [ABSTRACT FROM AUTHOR]

Rapid and effective diagnosis of communicable disease is one of the critical issues of the modern society, especially for detecting different targets at the same time. In this work, a grafting homogenous electrochemical biosensing strategy is proposed by integrating of reverse proximity ligation and exonuclease III (Exo III) assisted target circulation to analyze hepatitis B (HBV) and human immunodeficiency (HIV). Specially, a two-wing nanodevice (TWD) with two detection paths is elaborately designed based on analogous proximity ligation assay. The reverse proximity ligation process provides a new way of signal conversion and amplification, what accomplished by demolishing the TWD in the presence of targets. Meanwhile, a vast number of signal probes are released via Exo III assisted target circulation. Then the signal probes are grafted on the universal sensing interface, which is decorated with graftable tetrahedron DNA (GTD). These lead to a highly amplified electrochemical signal. Compared with the conventional strategies, the grafting homogenous electrochemical biosensing strategy not only achieves convenient sensitive detection of multiple communicable diseases DNA simultaneously, but also performs well in the detection of sole target. This strategy effectively decreases the background, homogenizes the distribution of probes, and avoids the complex and time-consuming modification process of the working electrode, which holds great potential application in early diagnosis for communicable disease in the future. • A two wing nanodevice is constructed by using the principle of analogous proximity ligation assay. • By using the principle of reverse proximity ligation assay, a new manner for conversing and amplifying the electrochemical signal is demonstrated for the first time. • The graftable tetrahedron DNA is immobilized on the electrode to construct a universal grafting sensing interface. • This strategy can be used to detect multiple targets simultaneously, and can also perform well in detection of sole target. [ABSTRACT FROM AUTHOR]