•Stable cross-linked DNAs were used for construction of bienzyme electrodes.•Glucose oxidase and horseradish peroxidase were immobilized on DNA scaffolds.•The detection sensitivity relied on the spatial positions of both enzymes.•The bienzyme/DNA complex could be also constructed on a microelectrode surface.•The bienzyme microelectrode allowed analyzing the distribution profile of glucose.Enzymes play an essential role in various detection technologies. We show here that interstrand cross-linked oligodeoxynucleotides (CL-ODNs) can provide stable scaffolds for efficiently coupling two types of enzymatic reactions on an electrode. Glucose can be electrochemically detected using glucose oxidase (GOx) and horseradish peroxidase (HRP). When both GOx and HRP were immobilized on an electrode surface by attachment at the termini of CL-ODNs, the current value was markedly increased compared with that obtained on a standard ODN scaffold. The relative orientation of the enzymes on the electrode strongly affected the current intensities. The CL-ODN also allowed GOx–HRP to form a complex on the tiny surface of a microelectrode, resulting in the imaging of local glucose distribution. These results suggest that CL-ODNs have potential utility in other sensing technologies.

•Nanoporous gold is formed on microelectrode by simple electrochemical treatment.•Bare gold microelectrodes are treated by oxidation reduction cycles in acidic media.•Large double-layer capacitance is observed in the voltammogram.•Surface area is 27 times higher than that of the bare electrode (Rf = 49).We fabricated nanoporous gold (NPG) on a microelectrode by oxidization and reduction cycles (ORCs) in acidic media. The typical morphology of the nanostructures comprised tangled thickets of branched nanowires with diameters of 20–30 nm. Furthermore, the surface area was significantly increased compared with the bare electrode. The roughness factor (Rf) of NPG could be controlled by changing the number of ORCs. The NPG-modified microelectrode with a roughness factor of 49 could immobilize 11 times more thiol-modified oligodeoxynucleotides on its surface than a microelectrode without NPG (Rf = 1.8). The results suggest that NPG formation is an important method for enhancing the electrochemical sensitivity of microelectrodes.Download high-res image (204KB)Download full-size image

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.

The electrochemically driven cytochrome P450 (CYP) reaction of a human microsome is expected to increase efficiency of drug metabolism assays and as well as prove useful for drug research. We previously reported that a nanostructured gold electrode modified with thiophenol (SPh) enabled the electrocatalytic CYP microsome reaction. However, repeated measurements resulted in a significant decrease in the activity. In the present study, we examined the immobilization and electrochemical measurements of the recombinant CYP2C9 microsome on gold electrodes modified with 4-aminothiophenol (SPh–NH2), 4-hydroxythiophenol, or 4-carboxythiophenol as the promoter. A clear pair of peaks in the voltammogram, assigned to the electron transfer between the electrode and CYP microsome, was observed at the SPh–NH2 modified surface. In the presence of oxygen and the well-known substrate, tolbutamide, the electrocatalytic current by the CYP reaction was observed. Interestingly, the responses were stable and were maintained compared with those at the SPh modified surface. It was suggested that this stable activity was related to less reactive oxygen species being produced at the SPh–NH2 modified surface. We also measured the tolbutamide metabolism reactions by the allelic variants of the CYP2C9 microsome on SPh–NH2 modified electrode. The estimated Km and kcat values were comparable to those obtained from the solution system. Therefore, SPh–NH2 modification gave an exquisite surface for electrochemically analyzing the recombinant CYP microsome reaction, indicating the usefulness for rapid assay of the enzyme.

We have synthesized a nonnucleoside amidite block of dansyl fluorophore to prepare dansyl-modified oligonucleotides (ONTs). The fluorescence intensities of dansyl-ONT specifically increased by the presence of adjacent guanosine residues but, significantly reduced in a dansyl-flipping duplex. These changes were caused by solvatochromism effect due to the number of guanine which is hydrophobic functional group and the external environment of dansyl group. The fluorescence intensities could be plotted as a function of the ONTs concentrations and the increase in the fluorescence was observed to equimolar concentrations of target DNA. This duplex exhibited higher melting temperature relative to the corresponding duplexes containing other base pairs. Similar changes in fluorescence could be detected upon hybridization with complementary RNAs. Thus, the dansyl-modified ONTs provide sequence specific fluorescent probe of DNA and RNA.

DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for double-stranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure.

Nanostructures of an electrode affect its functionality. We have developed a method for the fabrication of hexagonally ordered gold nanodome arrays with simple nanomorphology by employing the template-casting method. Templates with shallow concave shapes were fabricated using porous anodic aluminum oxide (AAO) and were then filled with gold. Scanning electron microscopy and atomic force microscopy images revealed that the morphology of the nanodomes was well controlled and uniformly shaped and had smooth hemispheres separated by steep valleys. Different anodization conditions gave different AAO interpore distances, enabling the generation of films having different cell diameters ranging over 27–109 nm. Furthermore, we confirmed that the gold nanodome arrays could function as electrodes for the direct electron transfer of cytochrome P450 immobilized on the electrode. This AAO-based electrode fabrication method provides functional electrodes, which are useful for studying the activation of redox enzymes and analyzing the functionality of nanostructures.Graphical abstractHighlights► We fabricated Au nanodome arrays by the template-casting method. ► The morphology of nanodomes was smooth hemispheres separated by steep valleys. ► The cell diameters of the nanodomes were in the range 27–109 nm. ► We achieved the direct electron transfer of P450 using the nanodome electrode.

We immobilized human cytochrome P450 (CYP), a membrane-bound enzyme, onto both smooth and nanostructured surfaces of gold electrodes via a naphthalene thiolate monolayer film. Rapid electron transfer of CYP with an electrode as a redox partner took place when the enzyme was immobilized onto an electrode surface with nanostructures. This structure was easily prepared by conventional sputtering techniques. A well-defined pair of peaks was observed at − 0.175 V (vs. SHE) with the largest heterogeneous electron transfer rate constant of 340 s− 1 for human CYP. The positive redox potential shift of 45 mV upon drug (testosterone) binding was clearly detected, which corresponded to a change in the spin states of heme iron in CYP. The present study showed that gold sputtered surfaces are very useful for direct electron transfer reactions of human CYP isoforms.

Abasic sites (AP sites) arise from hydrolysis of glycosidic bonds of DNA that is damaged by various external and internal processes; unrepaired AP sites give rise to genetic mutations. We have constructed highly reactive AP-site-detecting probes by introducing a hydrophobic and a hydrophilic residue in an aminooxy group. Synthesized probes containing either a naphthalene or a guanidine residue conjugate effectively with AP sites. In particular, a probe containing both functional groups shows the highest reaction rate, indicating that the hydrophobic and hydrophilic interactions act cooperatively in reaction with AP sites. The guanidine residue also contributes to the solubility of the molecules in aqueous media. The biotinylated probes provide much more sensitive detection of AP sites in genomic DNA than the conventional aldehyde-reactive probe.

Solid-support conjugation at the 5′-terminal primary amine of oligonucleotides is a convenient and powerful method for introducing various functional groups. However, conventional aliphatic amines do not necessarily provide conjugates with sufficient yields. To improve the modification efficacy, we used the amino-linker (aminoethoxycarbonyl)aminohexyl group (ssH-linker), for solid-support conjugation. In the ssH-linker terminal modification, reactive free amino group could be easily presented onto a solid-support due to rapid removal of the amino-protecting group, and activated amino acids or cholesterol molecules could be covalently connected more efficiently than to typical 6-aminohexyl-linkers. Based on these results, the ssH-linker can be a useful terminal modification for the solid-support conjugation of functional molecules.

Antifreeze proteins (AFPs) can protect cells from hypothermic damage; however, their mechanism of action remains unclear. Scanning electrochemical microscopy (SECM) can evaluate the size and activities of cells, although long-term continuous monitoring has been unsuccessful. We constructed a novel, fully automated, time-lapse SECM system and investigated the cell preservation effect of AFPs by analyzing single cellular topography at low temperatures. From the SECM measurements, mammalian cells (HepG2), treated in Euro-Collins (EC) solution at 4 °C, began to swell at 8 h and then immediately ruptured. In AFP-containing EC solution, the cellular size did not change until 16 h and then gradually increased and finally ruptured. In addition, the cellular height at rupture point significantly increased in the presence of AFPs. These results suggest that AFPs stabilize the cellular membrane and protect cells from hypothermic damage. This SECM system allowed us to observe the single cellular response to hypothermia by long-term automatic scanning and will be applicable for analysis to other cellular activities and topographies.

We developed new amino linker reagents for an oligonucleotide (ONT) terminus. These reagents consist of an aminoethyl carbamate main linkage and a side-chain residue, which was a naphthylmethoxymethyl, methoxymethyl, or methyl group or a hydrogen atom. The primary amine was protected with a monomethoxytrityl (MMT) group. The chemical properties of ONTs containing these amino-modifications were investigated. The MMT group of these amino-modifications could be quite rapidly removed from the amine under very mild acidic conditions, which are not strong enough for the deprotection of a conventional aliphatic amine. This significant feature enabled the amino-modified ONTs to be conveniently purified with a reverse phase column. Furthermore, the amino-modifications efficiently reacted to active esters, as compared with other amino-modifications. We also found that the pKa values of the amino-modifications were lower than that of the aliphatic amine. All of the experimental results showed that these chemical properties are closely related to their structures. We report here the chemical properties and the availability of the new amino linker reagents.The aminoethyl carbamate structure enabled the convenient purification of amino-modified oligonucleotides with a reverse phase column and increased the labeling efficiency with active esters in aqueous solution.

We developed novel amino-modifying reagents, of which an amino group was connected with an aromatic residue by aliphatic linker. It was proved that the insertion of the aromatic residue could increase the reactivity of the amino group on oligonucleotides in comparison with conventional amino-modification.

A pair of apurinic/apyrimidinic sites formed in DNA has been covalently connected with bis(aminooxy) derivatives. The efficacy of the interstrand cross-link is associated with the structural tethering of two aminooxy groups. The interstrand cross-link constructed stable DNA scaffolds for enzyme alignment.