Metal‐Ion‐Triggered Exonuclease III Activity for the Construction of DNA Colorimetric Logic Gates

The logic system is obtained by using a series of double‐stranded (ds) DNA templates with mismatched base pairs (T–T or C–C) and ion‐modulated exonuclease III (Exo III) activity, in which the Exo III cofactors, Hg²⁺ and Ag⁺ ions, are used as inputs for the activation of the respective scission of Exo III based on the formation of T–Hg²⁺–T or C–Ag⁺–C base pairs. Additionally, two kinds of signal probes are utilized to transduce the logic operations. One is the two split G‐rich DNA strands that are used to design the OR, AND, INHIBIT, and XOR gates, whereas the other is the self‐assembled split G‐quadruplex structure to construct NOR, NAND, IMPLICATION, and XNOR operations based on DNA hybridization and strand displacement. In the presence of hemin, the split G‐quadruplex biocatalyzes the formation of a colored product, which is an output signal for the different logic gates. Thus, we have constructed a complete set of colorimetric DNA logic gates based on the Exo III and split G‐quadruplex for the first time. In addition, we are able to effortlessly recognize the logic output signals by the naked eye and their simplicity and cost‐effective design is the most apparent feature for the logic gates developed in this work.     

A Reusable DNA Single‐Walled Carbon‐Nanotube‐Based Fluorescent Sensor for Highly Sensitive and Selective Detection of Ag+ and Cysteine in Aqueous Solutions

Here we report a reusable DNA single‐walled carbon nanotube (SWNT)‐based fluorescent sensor for highly sensitive and selective detection of Ag⁺ and cysteine (Cys) in aqueous solution. SWNTs can effectively quench the fluorescence of dye‐labeled single‐stranded DNA due to their strong π–π stacking interactions. However, upon incubation with Ag⁺, Ag⁺ can induce stable duplex formation mediated by C–Ag⁺–C (C=cytosine) coordination chemistry, which has been further confirmed by DNA melting studies. This weakens the interactions between DNA and SWNTs, and thus activates the sensor fluorescence. On the other hand, because Cys is a strong Ag⁺ binder, it can remove Ag⁺ from C–Ag⁺–C base pairs and deactivates the sensor fluorescence by rewrapping the dye‐labeled oligonucleotides around the SWNT. In this way, the fluorescence signal‐on and signal‐off of a DNA/SWNT sensor can be used to detect aqueous Ag⁺ and Cys, respectively. This sensing platform exhibits high sensitivity and selectivity toward Ag⁺ and Cys versus other metal ions and the other 19 natural amino acids, with a limit of detection of 1 nM for Ag⁺ and 9.5 nM for Cys. Based on these results, we have constructed a reusable fluorescent sensor by using the covalent‐linked SWNT–DNA conjugates according to the same sensing mechanism. There is no report on the use of SWNT–DNA assays for the detection of Ag⁺ and Cys. This assay is simple, effective, and reusable, and can in principle be used to detect other metal ions by substituting C–C base pairs with other native or artificial bases that selectively bind to other metal ions.     

G‐Quadruplex‐Linked Supersandwich DNA Structure for Electrochemical Amplified Detection of Thrombin

In this paper, a novel strategy of electrochemical amplified detection of thrombin based on G‐quadruplex‐linked supersandwich structure was described. In the presence of K⁺ and hemin, the original hairpin DNA sequence activated an autonomous cross‐opening process to build up hemin/G‐quadruplex structure and can hybridize to form supersandwich structure containing multiple signal labels. With the addition of thrombin, it conjugated with its aptamer, leading to a remarkably descended signal. The supersandwich‐amplified electrochemical sensor system was highly sensitive in the concentration range from 10^?6 to 10^?10 M with a detection limit of 10 pM and also demonstrated excellent selectivity. The amplifying supersandwich structure with multiple labels can be implemented as a versatile sensing platform for analyzing other DNA in the presence of the appropriate probe.     

Ion‐Responsive Hemin–G‐Quadruplexes for Switchable DNAzyme and Enzyme Functions

Programmed nucleic acid sequences undergo K⁺ ion‐induced self‐assembly into G‐quadruplexes and separation of the supramolecular structures by the elimination of K⁺ ions by crown ether or cryptand ion‐receptors. This process allows the switchable formation and dissociation of the respective G‐quadruplexes. The different G‐quadruplex structures bind hemin, and the resulting hemin–G‐quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K⁺ ion/receptor switchable systems are described: 1) The K⁺‐induced self‐assembly of the Mg²⁺‐dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G‐quadruplexes as bridging unit. 2) The K⁺‐induced stabilization of the anti‐thrombin G‐quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K⁺‐induced opening of DNA tweezers through the stabilization of G‐quadruplexes on the “tweezers’ arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin–G‐quadruplex DNAzyme).     

Orthogonal Dual‐Triggered Shape‐Memory DNA‐Based Hydrogels

DNA‐based shape‐memory hydrogels revealing switchable shape recovery in the presence of two orthogonal triggers are described. In one system, a shaped DNA/acrylamide hydrogel is stabilized by duplex nucleic acids and pH‐responsive cytosine‐rich, i‐motif, bridges. Separation of the i‐motif bridges at pH 7.4 transforms the hydrogel into a quasi‐liquid, shapeless state, that includes the duplex bridges as permanent shape‐memory elements. Subjecting the quasi‐liquid state to pH 5.0 or Ag⁺ ions recovers the hydrogel shape, due to the stabilization of the hydrogel by i‐motif or C‐Ag⁺‐C bridged i‐motif. The cysteamine‐induced transformation of the duplex/C‐Ag⁺‐C bridged i‐motif hydrogel into a quasi‐liquid shapeless state results in the recovery of the shaped hydrogel in the presence of H⁺ or Ag⁺ ions as triggers. In a second system, a shaped DNA/acrylamide hydrogel is generated by DNA duplexes and bridging Pb²⁺ or Sr²⁺ ions‐stabilized G‐quadruplex subunits. Subjecting the shaped hydrogel to the DOTA or KP ligands eliminates the Pb²⁺ or Sr²⁺ ions from the respective hydrogels, leading to shapeless, memory‐containing, quasi‐liquid states that restore the original shapes with Pb²⁺ or Sr²⁺ ions.     

Elongated Thrombin Binding Aptamer: A G‐Quadruplex Cation‐Sensitive Conformational Switch

Aptamer‐based biosensors offer promising perspectives for high performance, specific detection of proteins. The thrombin binding aptamer (TBA) is a G‐quadruplex‐forming DNA sequence, which is frequently elongated at one end to increase its analytical performances in a biosensor configuration. Herein, we investigate how the elongation of TBA at its 5′ end affects its structure and stability. Circular dichroism spectroscopy shows that TBA folds in an antiparallel G‐quadruplex conformation with all studied cations (Ba²⁺, Ca²⁺, K⁺, Mg²⁺, Na⁺, NH₄⁺, Sr²⁺ and the [Ru(NH₃)₆]^2+/3+ redox marker) whereas other structures are adopted by the elongated aptamers in the presence of some of these cations. The stability of each structure is evaluated on the basis of UV spectroscopy melting curves. Thermal difference spectra confirm the quadruplex character of all conformations. The elongated sequences can adopt a parallel or an antiparallel structure, depending on the nature of the cation; this can potentially confer an ion‐sensitive switch behavior. This switch property is demonstrated with the frequently employed redox complex [Ru(NH₃)₆]³⁺, which induces the parallel conformation at very low concentrations (10 equiv per strand). The addition of large amounts of K⁺ reverts the conformation to the antiparallel form, and opens interesting perspectives for electrochemical biosensing or redox‐active responsive devices.     

Dual Amplified Electrochemical Immunosensor for Hepatitis B Virus Surface Antigen Detection Using Hemin/G‐Quadruplex Immobilized onto Fe3O4‐AuNPs or (Hemin‐Amino‐rGO‐Au) Nanohybrid

A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe₃O₄‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe₃O₄‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H₂O₂ in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe₃O₄‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results.     

Surface plasmon resonance detection of silver ions and cysteine using DNA intercalator-based amplification

We report the development of a surface plasmon resonance sensor based on the silver ion (Ag⁺)-induced conformational change of a cytosine-rich, single-stranded DNA for the detection of Ag⁺ and cysteine (Cys) in aqueous solutions. In the free state, single-stranded oligonucleotides fold into double-helical structures through the addition of Ag⁺ to cytosine–cytosine (C–C) mismatches. However, in the presence of Cys, which competitively binds to Ag⁺, the formation of the C–Ag⁺–C assembly is inhibited, resulting in free-state, single-stranded oligonucleotides. To enhance sensitivity, the DNA intercalator, daunorubicin, was employed to achieve signal enhancement. The detection limit for Ag⁺ was 10 nM with a measurement range of 50–2,000 nM, and the detection limit for Cys was 50 nM with a measurement range of 50–2,000 nM. This simple assay was also used to individually determine the spiked Ag⁺ concentration in water samples and Cys concentrations in biological fluid samples.     

Ion‐Selective Formation of a Guanine Quadruplex on DNA Origami Structures

DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single‐molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G‐quadruplex structures in the presence of monovalent cations (e.g. Na⁺ and K⁺) is currently used for the detection of K⁺ ions, however, with insufficient selectivity towards Na⁺. By means of FRET between two suitable dyes attached to the 3′‐ and 5′‐ends of telomeric DNA we demonstrate that the formation of G‐quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.     

Detection of Metal Ions (Cu2+, Hg2+) and Cocaine by Using Ligation DNAzyme Machinery

The Cu²⁺‐dependent ligation DNAzyme is implemented as a biocatalyst for the colorimetric or chemiluminescence detection of Cu²⁺ ions, Hg²⁺ ions, or cocaine. These sensing platforms are based on the structural tailoring of the sequence of the Cu²⁺‐dependent ligation DNAzyme for specific analytes. The tethering of a subunit of the hemin/G‐quadruplex DNAzyme to the ligation DNAzyme sequence, and the incorporation of an imidazole‐functionalized nucleic‐acid sequence, which acts as a co‐substrate for the ligation DNAzyme that is tethered to the complementary hemin/G‐quadruplex subunit. In the presence of different analytes, Cu²⁺ ions, Hg²⁺ ions, or cocaine, the pretailored Cu²⁺‐dependent ligation DNAzyme sequence stimulates the respective ligation process by combining the imidazole‐functionalized co‐substrate with the ligation DNAzyme sequence. These reactions lead to the self‐assembly of stable hemin/G‐quadruplex DNAzyme nanostructures that enable the colorimetric analysis of the substrate through the DNAzyme‐catalyzed oxidation of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid), ABTS^2?, by H₂O₂ into the colored product ABTS^.?, or the chemiluminescence detection of the substrate through the DNAzyme‐catalyzed oxidation of luminol by H₂O₂. The detection limits for the sensing of Cu²⁺ ions, Hg²⁺ ions, and cocaine correspond to 1 nM , 10 nM and 2.5 μM , respectively. These different sensing platforms also reveal impressive selectivities.     

Modelling Photosynthesis with ZnII‐Protoporphyrin All‐DNA G‐Quadruplex/Aptamer Scaffolds

All‐DNA scaffolds act as templates for the organization of photosystem I model systems. A series of DNA templates composed of Zn^II‐protoporphyrin IX (Zn^IIPPIX)‐functionalized G‐quadruplex conjugated to the 3′‐ or 5′‐end of the tyrosinamide (TA) aptamer and Zn^IIPPIX/G‐quadruplex linked to the 3′‐ and 5′‐ends of the TA aptamer through a four‐thymidine bridge. Effective photoinduced electron transfer (ET) from Zn^IIPPIX/G‐quadruplex to bipyridinium‐functionalized tyrosinamide, TA‐MV²⁺, bound to the TA aptamer units is demonstrated. The effectiveness of the primary ET quenching of Zn^IIPPIX/G‐quadruplex by TA‐MV²⁺ controls the efficiency of the generation of TA‐MV^+.. The photosystem‐controlled formation of TA‐MV^+. by the different photosystems dictates the secondary activation of the ET cascade corresponding to the ferredoxin‐NADP⁺ reductase (FNR)‐catalysed reduction of NADP⁺ to NADPH by TA‐MV^+., and the sequestered alcohol dehydrogenase catalysed reduction of acetophenone to 1‐phenylethanol by NADPH.     

Switchable Enzyme/DNAzyme Cascades by the Reconfiguration of DNA Nanostructures

Mimicking cellular transformations and signal transduction pathways by means of biocatalytic cascades proceeding in organized media is a scientific challenge. We describe two DNA machines that enable the “ON/OFF” switchable activation and deactivation of three‐component biocatalytic cascades. One system consists of a reconfigurable DNA tweezers‐type structure, whereas in the second system the catalytic cascade proceeds on a switchable DNA clamp scaffold. The three‐component catalytic cascades consist of β‐galactosidase (β‐Gal), glucose oxidase (GOx), and the K⁺‐ion‐stabilized hemin‐G‐quadruplex horseradish peroxidase (HRP)‐mimicking DNAzyme. The hemin‐G‐quadruplex‐bridged closed structure of the tweezers or clamp allows the biocatalytic cascades to operate (switched “ON′′), whereas separation of the hemin‐G‐quadruplex by means of 18‐crown‐6‐ether opens the tweezers/clamp structures, thus blocking the catalytic cascade (switched ”OFF“). This study is complemented by two‐component, switchable biocatalytic cascades composed of GOx and hemin‐G‐quadruplex assembled on hairpin‐bridged DNA tweezers or clamp nanostructures.     

G‐Quadruplex‐Based DNAzymes Aptasensor for the Amplified Electrochemical Detection of Thrombin

A novel G‐quadruplex‐based DNAzymes aptasensor for the amplified electrochemical detection of thrombin has been described. The aptasensor utilized a combination of hemin and guanine‐rich thrombin‐binding aptamer (TBA) to form horseradish peroxidase (HRP)‐mimicking DNAzymes with peroxidase catalytic activity. In the presence of thrombin, the enzyme activity could be extensively promoted, thereby providing the amplified electrochemical readout signals for detecting thrombin. This aptasensor exhibited high sensitivity and selectivity for thrombin determination, which enabled the analysis of thrombin with a detection limit of 6×10^–11 M. On the basis of results, this method could have broad applications in the detection of proteins and other biomolecules.     

Structure‐Based Design of Platinum(II) Complexes as c‐myc Oncogene Down‐Regulators and Luminescent Probes for G‐Quadruplex DNA

A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G‐quadruplex DNA within the c‐myc gene promoter were evaluated. Complex 1 , which has a flat planar 2,6‐bis(benzimidazol‐2‐yl)pyridine (bzimpy) scaffold, was found to stabilize the c‐myc G‐quadruplex structure in a cell‐free system. An in silico G‐quadruplex DNA model has been constructed for structure‐based virtual screening to develop new Pt^II‐based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit‐to‐lead optimization, bzimpy and related 2,6‐bis(pyrazol‐3‐yl)pyridine (dPzPy) scaffolds containing amine side‐chains emerge as the top candidates. Six of the top‐scoring complexes were synthesized and their interactions with c‐myc G‐quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c‐myc G‐quadruplex. Complex 3 a ([Pt^II L2R ] ⁺ ; L2 =2,6‐bis[1‐(3‐piperidinepropyl)‐1H‐enzo[d]imidazol‐2‐yl]pyridine, R =Cl) displayed the strongest inhibition in a cell‐free system (IC₅₀=2.2 μM ) and was 3.3‐fold more potent than that of 1 . Complexes 3 a and 4 a ([Pt^II L3R ]⁺; L3 =2,6‐bis[1‐(3‐morpholinopropyl)‐1H‐pyrazol‐3‐yl]pyridine, R =Cl) were found to effectively inhibit c‐myc gene expression in human hepatocarcinoma cells with IC₅₀ values of ≈17 μM , whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 μM . Complexes 3 a and 4 a have a strong preference for G‐quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G‐quadruplex DNA with binding constants (K) of approximately 10⁶–10⁷ dm³ mol^?1, which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3′‐terminal face of the G‐quadruplex. Intriguingly, binding of c‐myc G‐quadruplex DNA by 3 b is accompanied by an increase of up to 38‐fold in photoluminescence intensity at λ_max=622 nm.     

Fluorescent detection of silver(I) and cysteine using SYBR Green I and a silver(I)-specific oligonucleotide

We report on a novel method for the determination of silver ion (Ag⁺) and cysteine (Cys) by using the probe SYBR Green I (SGI) and an Ag+-specific cytosine-rich oligonucleotide (C-DNA). The fluorescence of SGI is very weak in the absence or presence of randomly coiled C-DNA. If, however, C-DNA interacts with Ag⁺ through the formation of cytosine-Ag⁺-cytosine (C-Ag⁺-C) base pairs, the randomly coiled C-DNA undergoes a structural changes to form a hairpin-like structure, thereby increasing the fluorescence of SGI. This fluorescence turn-on process allows the detection of Ag⁺ in the 10–600?nM concentration range, with a detection limit of 4.3?nM. Upon the reaction of Ag⁺ with Cys, Cys specifically removes Ag⁺ from the C-Ag⁺-C base pairs and destroys the hairpin-like structure. This, in turn, results in a decrease in fluorescence intensity. This fluorescence turn-off process enables the determination of Cys in the 8–550?nM concentration range, with a detection limit of 4.5?nM. The method reported here for the determination of either Ag⁺ or Cys is simple, sensitive, and affordable, and may be applied to other detection systems if appropriately selected DNA sequences are available.

Binding Behaviors for Different Types of DNA G‐Quadruplexes: Enantiomers of [Ru(bpy)2(L)]2+ (L=dppz,dppz‐idzo)

Polymorphic DNA G‐quadruplex recognition has attracted great interest in recent years. The strong binding affinity and potential enantioselectivity of chiral [Ru(bpy)₂(L)]²⁺ (L=dipyrido[3,2‐a:2′,3′‐c]phenazine, dppz‐10,11‐imidazolone; bpy=2,2′‐bipyridine) prompted this investigation as to whether the two enantiomers, Δ and Λ, can show different effects on diverse structures with a range of parallel, antiparallel and mixed parallel/antiparallel G‐quadruplexes. These studies provide a striking example of chiral‐selective recognition of DNA G‐quadruplexes. As for antiparallel (tel‐Na⁺) basket G‐quadruplex, the Λ enantiomers bind stronger than the Δ enantiomers. Moreover, the behavior reported here for both enantiomers stands in sharp contrast to B‐DNA binding. The chiral selectivity toward mixed parallel/antiparallel (tel‐K⁺) G‐quadruplex of both compounds is weak. Different loop arrangements can change chiral complex selectivity for both antiparallel and mixed parallel/antiparallel G‐quadruplex. Whereas both Δ and Λ isomers bind to parallel G‐quadruplexes with comparable affinity, no appreciable stereoselective G‐quadruplex binding of the isomers was observed. In addition, different binding stoichiometries and binding modes for Δ and Λ enantiomers were confirmed. The results presented here indicate that chiral selective G‐quadruplex binding is not only related to G‐quadruplex topology, but also to the sequence and the loop constitution.     

A Highly Stabilizing Silver(I)‐Mediated Base Pair in Parallel‐Stranded DNA

The first parallel‐stranded DNA duplex with Hoogsteen base pairing that readily incorporates an Ag⁺ ion into an internal mispair to form a metal‐mediated base pair has been created. Towards this end, the highly stabilizing 6 FP ‐Ag⁺‐ 6 FP base pair comprising the artificial nucleobase 6‐furylpurine ( 6 FP ) was devised. A combination of temperature‐dependent UV spectroscopy, CD spectroscopy, and DFT calculations was used to confirm the formation of this base pair. The nucleobase 6 FP is capable of forming metal‐mediated base pairs both by the Watson–Crick edge (i.e. in regular antiparallel‐stranded DNA) and by the Hoogsteen edge (i.e. in parallel‐stranded DNA), depending on the oligonucleotide sequence and the experimental conditions. The 6 FP ‐Ag⁺‐ 6 FP base pair within parallel‐stranded DNA is the most strongly stabilizing Ag⁺‐mediated base pair reported to date for any type of nucleic acid, with an increase in melting temperature of almost 15 °C upon the binding of one Ag⁺ ion.     

A G‐Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

A hemin‐binding DNA G‐quadruplex (also known as a hemin aptamer or DNAzyme) has been previously reported to be able to enhance the peroxidase activity of hemin. In this work, we described a DNAzyme structure that had an effector‐recognizing part appearing as a single stranded DNA linkage flanked by two split G‐quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G‐quadruplex halves and thus efficiently suppressed the enzymatic activity of the G‐quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2′‐azinobis(3‐ethylbenzthiazoline)‐6‐sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re‐engineered G‐quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G‐quadruplex is an especially useful module to design low‐cost and label‐free sensors toward various biologically or environmentally interesting targets.     

A Highly Sensitive Electrochemiluminescence Choline Biosensor Based on Poly(aniline‐luminol‐hemin) Nanocomposites

Poly(aniline‐luminol‐hemin) nanocomposites are prepared on an electrode surface through electropolymerization, and a highly sensitive electrochemiluminescence (ECL) biosensor for choline is developed based on the poly(aniline‐luminol‐hemin) nanocomposites and an enzyme catalyzed reaction of choline oxidase (CHOD). The obtained nanocomposites are characterized by scanning electron microscopy (SEM), atomic absorption spectrometry (AAS) and ECL. The results indicate that hemin can be incorporated into the poly(aniline‐luminol) nanocomposites using the facile electropolymerization method, and the poly(aniline‐luminol‐hemin) nanocomposites are rod shaped porous nanostructure. Moreover, the poly(aniline‐luminol‐hemin) nanocomposites exhibit higher ECL intensity than poly(aniline‐luminol) nanocomposites in alkaline media due to the catalytic effect of hemin on the ECL of the polymerized luminol and the electron transfer ability of hemin in the nanocomposites. CHOD is immobilized on the surface of the poly(aniline‐luminol‐hemin) nanocomposites modified electrode with glutaraldehyde, and the ECL biosensor based on poly(aniline‐luminol‐hemin)/CHOD exhibits a wider linear range for the choline detection. The enhanced ECL signals are linear with the logarithm of concentration of choline over the range of 1.0×10^?11～1.0×10^?7 mol L^?1 with a low detection limit of 1.2×10^?12 mol L^?1. Moreover, the proposed biosensor is successfully applied to the detection of choline in milk.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.