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 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.

Reactivity of ferrocenylcarbodiimide with DNA duplex containing single-mismatched base pairs.

Ferrocenylcarbodiimide (1), which is known to react with a guanine (G) or thymine (T) base of single stranded DNA, was allowed to react with DNA duplex having a single mismatched base pair of G-T, T-T, or T-cytosine (C). Electrophoreograms of the reaction mixture showed that 1 could react with G or T base of the mismatched sites on the DNA duplex. However, 1 also reacted with the G base of the terminal site on the DNA duplex. This showed that 1 can react with an unpaired base or unstable base pair such as a terminal or mismatched base on the DNA duplex. Electrochemical mismatch detection could be achieved after hybridization of the ferrocenylated mismatched DNA duplex with a selected DNA probe-immobilized electrode. These results revealed that 1 has a potentiality of serving as a labeling reagent of mismatched bases on the DNA duplex, which is important in the search for heterozygous single nucleotide polymorphisms (SNPs).

Direct modification of mRNA by ferrocenyl carbodiimide and its application to electrochemical detection of mRNA.

Ferrocenyl carbodiimide (1) could be used for the direct labeling of synthetic RNA and expressed mRNA in vitro with the electrochemically active ferrocene moieties. These RNAs modified by 1 could be detected electrochemically coupled with a DNA probe-immobilized electrode. After hybridization of 1.1 Kb mRNA modified by 1 with the DNA probe-immobilized electrode, the peak charge observed by an Osteryoung square wave voltammetry (SWV) measurement correlated well with the concentration of mRNA, having a detection limit at the sub nanogram level.

Electrochemical detection of DNase I activity.

DNase I in one microl of the water could quantitate electrochemically with the detection limit of 0.01 units (ca. 20 pg) by using the ferrocenyl oligonucleotide-immobilized electrode prepared by thiolated oligonucleotide and ferrocenyl carbodiimide as a simple labeling reagent of redox unit.

Electrochemical SNP detection.

Detection of mismatched base on a DNA duplex, which is important for the search of the heterozygote, achieved by the comparison with the reactivity of ferrocenylcarbodiimide (1) developed by our group. Mismatched thymine base on the 30-meric DNA duplex as a model of SNPs in lipoprotein lipase (LPL) gene, could react with 1 in 20 mM borate buffer (pH 8.5) and 0.1 M NaCl containing 20% DMSO at 37 degrees C. DNA duplex modified by 1 in the mismatched thymine bases could be detected by its shifted migration time in microTAS electrophoresis.

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.

Ferrocenylnaphthalene diimide-based electrochemical detection of methylated gene.

Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16(Ink4a)) were used to be immobilized on the electrochemical array (ECA) chip. Using 1 microL of 10 ng/microL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.

Interaction analysis of the carcinoembryonic antigen (CEA) with its monoclonal antibody immobilized on a gold surface using Fourier transform infrared reflection-absorption spectroscopy (FT-IR RAS).

A monoclonal antibody for the carcinoembryonic antigen (CEA) was immobilized on a gold chip surface covered by a self-assembled monolayer of 11-mercaptoundecanoic acid. Upon the addition of CEA, a Fourier transform infrared reflection-absorption spectroscopy (FT-IR RAS) measurement showed an increased absorption at around 1500 - 1700 cm(-1), corresponding to its amide structures. Another addition of CEA polyclonal antibody on this chip caused a further increase of the absorption in this region only after a treatment with CEA. This result shows that an antibody-fixed gold surface coupled with an FT-IR RAS measurement provides a new tool for detecting the antibody-antigen interaction.

Fluorescence enhancement of bis-acridine orange peptide, BAO, upon binding to double stranded DNA.

Bis-acridine orange peptides carrying two acridine oranges at the epsilon-amino moieties of both terminal lysines of a tetra(lysine) chain showed a ca. 200-fold fluorescence enhancement upon addition of double stranded DNA.

Supramolecular complex formation by beta-cyclodextrin and ferrocenylnaphthalene diimide-intercalated double stranded DNA and improved electrochemical gene detection.

Ferrocenylnaphthalene diimide 1 can bind to double stranded DNA (dsDNA) by the threading intercalation mode and the resulting complex was stabilized further by beta- cyclodextrin (CD) by forming a supramolecular complex. These complex formation processes were studied by spectroscopic, viscometric, and electrochemical means in the absence or presence of beta-CD. Quantitative analysis by quartz crystal microbalance (QCM) and electrochemical experiments strongly suggested a 2:1 binding stoichiometry for beta-CD to 1 threading-intercalated to the dsDNA-immobilized electrode. Owing to this supramolecular complex formation, electrochemical DNA detection based on 1 was improved considerably.

Immobilization of sunflower trypsin inhibitor (SFTI-1) peptide onto a gold surface and analysis of its interaction with trypsin.

Sunflower trypsin inhibitor (SFTI-1) derived peptide having one disulfide bond could be immobilized via a thiol-disulfide exchange reaction onto a gold surface on a quartz crystal microbalance (QCM) chip. This permitted quantitative analysis of the specific interaction with trypsin.