Keiichi Ohtsuka

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k(cat)/K(m) value was 0.063 microM(-1) s(-1). Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.

Electrochemical DNA Analysis with a Supramolecular Assembly of Naphthalene Diimide, Ferrocene, and β-Cyclodextrin

Naphthalene diimide 1 bearing ferrocene and β-cyclodextrin (β-CD) was prepared. Its half-wave potential at 420 mV shifted 40-50 mV upon addition of an excess of adamantylamine, suggesting that the ferrocene of 1 is included in the cavity of β-CD intramolecularly to form a pseudocyclic structure. This unique architecture is retained even where 1 is bound to calf thymus DNA to give rise to a catenane-like structure. Morphology of the DNA complex with 1 was further explored by atomic force microscopy to reveal that the DNA strand tends to bend as the amount of 1 on it increases. Presumably, intermolecular, yet intrastrand, inclusion of ferrocene into β-CD is responsible for this phenomenon. The resulting globular structure reverted partially by the addition of adamantylamine. At a low ratio of 1 to DNA, a novel reduction peak appeared at 320 mV in the differential pulse voltammograms of 1 at the expense of the 420 mV peak. The peak current of the former was proportional to the DNA concentration, thereby enabling quantitation of DNA in a signal-on way. Likewise, a polymerase chain reaction (PCR) product of 754 bp was analyzed successfully with a detection limit of 13 nM.

Reliable ferrocenyloligonucleotide-immobilized electrodes and their application to electrochemical DNase I assay

A ferrocenyloligonucleotide (FcODN) having contiguous cytosine bases was immobilized effectively and reproducibly on a gold electrode furnished with a self-assembled monolayer (SAM) having an N-hydroxysuccinimide-activated carboxylic acid. The resulting electrode was used as a sensor chip in DNase I assay. Thus, the current response of the modified electrode decreased upon addition of DNase I, demonstrating that the phosphodiester bonds of FcODN were cleaved. The DNase I activity assessed by Deltai defined as (i0-i)/i0, where i0 and i represent the current before and after DNase I treatment, respectively, was found to be reproducible with a standard deviation not greater than 9%. The DNase I can be quantitated in the range of 10(-5) to 10(-3) units microL(-1) with a detection limit of 10(-5) units microL(-1) with this sensor chip. The current signal of the FcODN electrode was stable to storage in Biopak water up to 16 days with a 30% signal decrease over this period. DNase I activity in human sera was also determined successfully with this sensor chip.

Discrimination of phosphorylated double stranded DNA by naphthalene diimide having zinc(II) dipicolylamine complexes

Discrimination of phosphomonoesters and phosphodiesters of DNA was attempted with naphthalene diimide carrying two zinc-dipicolylamine (Dpa) units (1). The binding constant of 1 for a self-complementary octanucleotide was 1.3×10(6)M(-1), while the value for the phosphorylated counterpart was 4.8×10(6)M(-1). This fourfold increase in the binding constant seems to stem from higher affinity of the terminal monophosphate over the phosphodiesters of DNA as the fourth ligand for the metal in 1. Likewise, the binding constant of 1 for DNase I-treated calf thymus DNA (average size 200bp) was twice as large as that for untreated DNA (1kb), possibly because the terminal phosphate groups are five times abundant in the former. These findings provide a clue to developing a system where phosphomonoesters generated upon DNA nicking are discriminated specifically from intact phosphodiesters.

Electrochemical assay for deoxyribonuclease I activity

A thiolated oligonucleotide having three ferrocenes was immobilized on a gold electrode through the sulfur-gold linkage. This electrode showed a current response based on the redox reaction of the ferrocene moieties and this response was decreased after treatment with deoxyribonuclease I (DNase I), suggesting the disappearance of the ferrocene moieties on the electrode by the DNase I digestion. A linear correlation between i(0) and i, which are current peaks before and after DNase I treatment, respectively, was observed and this slope was decreased with increase in the amount of DNase I. No current decrease was observed in the presence of EDTA or RNase A instead of DNase I. These results suggested that the current decrease responded specifically to the amount of DNase I and this electrode could be used for an electrochemical DNase I assay. Under the optimum conditions of DNase I digestion at 37 degrees C for 30 min, a quantitative analysis could be achieved in the range of 10(-4)-10(-2)units/microl of DNase I.

Immobilization of RNase S-Peptide on a single-stranded DNA-fixed gold surface and effective masking of its surface by an acridinyl poly(ethylene glycol)

Oligonucleotide-peptide conjugate was synthesized by coupling of RNase S-peptide to a 24-mer single-stranded DNA (ssDNA) oligonucleotide to be immobilized on its complementary ssDNA oligonucleotide-fixed gold surface of sensor chip or electrode. Immobilization of on the ssDNA-fixed gold surface through DNA duplex formation was confirmed by quartz crystal microbalance (QCM) and electrochemical measurements. After treating with a synthetic acridinyl poly(ethylene glycol) (APEG), specific interaction of S-protein with the S-peptide immobilized on the gold surface was demonstrated by QCM without nonspecific adsorption of unrelated proteins such as BSA and RNase A at the surfaces. This result suggested that the acridine parts of APEG could bind to the DNA duplex on the gold surface and the poly(ethylene glycol) parts were fastened on the surface to resist the adsorption of proteins. Thus, the combination of oligonucleotide-peptide conjugate, ssDNA-fixed chip and APEG with effective masking property provides a new tool for the analysis of specific peptide-protein interactions without disturbance by other unrelated proteins.

Synthesis of a naphthalene diimide derivative having four ferrocene moieties as an electrochemical DNA hybridization indicator

A new naphthalene diimide derivative carrying four ferrocene moieties, F(4)ND, was synthesized by the reaction of trimethylammoniummethylferrocene with the terminal amino moieties of two imide substituents of naphthalene diimide. Spectrophotometric binding studies of F(4)ND with calf thymus DNA showed its high affinity of 2 x 10(5) M(-1) in 10 mM MES buffer and 1 mM EDTA (pH 6.25) containing 0.10 M NaCl. The Osteryoung square wave voltammetry (SWV) of a DNA probe-immobilized electrode before and after hybridization with target DNA gave a larger peak current in an electrolyte containing F(4)ND than that containing FND carrying two ferrocene moieties. The current peak difference between mismatched and fully matched DNA was also greater with F(4)ND than that with FND.

Immobilization of a naphthalene diimide-DNA complex on the gold through dithiolane moieties

Naphthalene diimide 1 having two dithiolane moieties at its substituted termini was newly synthesized to immobilize double stranded DNA through dithiolane moieties of 1. Double stranded DNA could be immobilized on the gold surface of a quartz crystal microbalance (QCM) chip after binding 1. Once immobilized, the complex will serve as a hybridization marker based on its frequency decrease. QCM experiments showed that a DNA duplex with a 24-meric single stranded region also could be immobilized on the gold surface of a QCM chip and subsequent frequency decrease was observed after hybridization with a 24-meric complementary DNA, but not with non-complementary DNA, indicating specific hybridization. This result reveals that 1 provides a new immobilization method for intact DNA on gold and that the immobilized DNA can hybridize with target DNA.

Synthesis of naphthalenediimide having two β-cyclodextrins at both of its substituent termini

Naphthalenediimide (1) carrying two beta-cyclodextrins (beta CDs) was successfully synthesized by click chemistry between pentynylnaphthalenediimide and azido beta CD. Absorption spectra of 1 showed hypochromic and red shifts upon addition of sonicated calf thymus DNA as a double stranded DNA. Kinetic studies showed its slow association with and dissociation from calf thymus DNA. These results suggested that 1 can bind to double stranded DNA by the threading mode, where one of the beta CD is required to go through adjacent base pairs of double stranded DNA. If this is proven, beta CD is one of the largest groups ever reported which can go through a DNA duplex for threading.

DNA methylation detection based on difference of base content

Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.