Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film.

We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp(2) and sp(3) mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (approximately 24 mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 5'-cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60 mers) with different methylation ratios.

Local imaging of an electrochemical active/inactive region on a conductive carbon surface by using scanning electrochemical microscopy.

We demonstrated the imaging of local electron transfer-rate differences on a flat conductive carbon substrate, attributed to only surface functional groups, by using a scanning electrochemical microscopy (SECM) technique. These differences were clearly imaged by using a redox mediator with surface state sensitive electron transfer rates, even if the conductivity of each imaging area were almost identical. The carbon electrode surface was masked with a patterned photoresist, and selectively introduced oxygen functional groups using an oxygen plasma treatment. This patterned surface exhibited hardly any topographical features when observed by scanning electron microscopy (SEM), and a height difference of only 1.0 nm was observed with atomic force microscopy (AFM). However, the SECM feedback mode and substrate generation-tip collection (SG-TC) mode are able to distinguish these interfaces with an almost micrometer order resolution by utilizing the difference in the electron transfer rate for the Fe(2+/3+) mediator, and the current ratios for regions rich and poor in oxygen containing groups were 1.5 and 2.0, respectively. This technique could be employed for imaging and monitoring the electron transfer rates on various electrode surfaces, including fluorine and nitrogen terminated surfaces and a monolayer film patterned with micro or nano contact printing techniques.

Bioelectrochemical conversion of urea to nitrogen using aminated carbon electrode.

Urea decomposes to ammonia and carbon dioxide via carbamic acid, and amine groups can be introduced to the glassy carbon electrode surface during the electrode oxidation of carbamic acid. This modified carbon electrode has excellent catalytic activity of the oxidation of carbamic acid, and can be used to electrooxidize urea by combining urease reaction and electrode oxidation. We found that nitrogen gas is finally produced by the carbamic acid produced from urea. The production of nitrogen was confirmed by gas chromatography-mass spectrometry, and fragment pattern of hydrazine was also detected in the electrolyzed solution of urea. We intend to describe new electrochemical conversion system of urea to harmless nitrogen gas. The electrode oxidation current of urea was decreased by addition of radical trapping agent such as DMPO (5,5-dimethyl-1-pyrroline N-oxide), and this fact suggests that carbamic acid radical couples to form nitrogen-nitrogen bond, and this dimer is oxidized to nitrogen. The electrode oxidation current of urea became larger when oxygen was removed. This fact indicates that the intermediate species (probably hydrazine) produced by the electrolysis is oxidized by not only electrode reaction but also oxygen.

A nanocarbon film electrode as a platform for exploring DNA methylation.

We describe the quantitative nonlabel electrochemical detection of both cytosine (C) and methylcytosine (mC) in oligonucleotides using newly developed nanocarbon film electrodes. The film consists of nanocrystalline sp2 and sp3 mixed bonds formed by employing the electron cyclotron resonance (ECR) sputtering method. We successfully used this film to develop a simple electrochemical DNA methylation analysis technique based on the measurement of the differences between the oxidation currents of C and mC since our ECR nanocarbon film electrode can directly measure all DNA bases more quantitatively than conventional glassy carbon or boron-doped diamond electrodes. The excellent properties of ECR nanocarbon film electrodes result from the fact that they have a wide potential window while maintaining the high electrode activity needed to oxidize oligonucleotides electrochemically. Proof-of-concept experiments were performed with synthetic oligonucleotides including different numbers of C and mC. This film allowed us to perform both C- and mC-positive assays solely by using the electrochemical oxidation of oligonucleotides without bisulfite or labeling processes.

Application of polymaleimidostyrene as a convenient immobilization reagent of enzyme in biosensor.

Sulfhydryl groups of glucose oxidase (GOD) were reacted with maleimide groups of polymaleimidostyrene (PMS) which was coated onto the porous carbon sheet, and the carbon sheet immobilized by GOD was combined with an oxygen electrode to fabricate a glucose sensor. The activity of thiolated GOD immobilized to PMS is much larger than that of native GOD immobilized to PMS. The good linear relationship of glucose and oxygen current response was obtained in a concentration range from 0.1 to 2 mM and upper limit of linear range was found to be 3.0 mM. The immobilized GOD activity is highly dependent on pH at immobilization and the maximum activity was obtained at pH 5.5, probably because the SH groups of GOD that are indispensable for generation of enzyme activity is not exposed at this pH. It was found that PMS is very effective reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups.