Fluorescence imaging of potassium ions in living cells using a fluorescent probe based on a thrombin binding aptamer-peptide conjugate.

When a biotinylated FRET probe based on a peptide-thrombin binding aptamer conjugate was introduced together with streptavidin and biotinylated nuclear export signal peptide into HeLa cells, the resulting ternary complex enabled visualization of K(+) concentration changes in the cell.

Electrochemical DNA analysis with a supramolecular assembly of naphthalene diimide, ferrocene, and ß-cyclodextrin.

Naphthalene diimide 1 bearing ferrocene and ß-cyclodextrin (ß-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 ß-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 ß-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.

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.

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.

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.

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.

Detection of an antibody to avian influenza virus by an electrochemical immunoassay (eELISA).

The detection of an antibody against nucleoprotein (NP) of the avian influenza virus (AIV) was attempted by an electrochemical immunoassay by combining a secondary antibody-alkaline phosphatase conjugate and 4-aminophenylphosphate. Released 4-aminophenol was quantitated electrochemically, and the current derived from oxidation of the hydrolysis product increased linearly over a wide primary antibody concentration range (10 - 1000 ng/ml). The detection limit of this electrochemical immunoassay for the antibody against the NP of AIV was found to be 10 ng/ml.

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.

Synthesis of a perylene diimide derivative having two ferrocene moieties as an electrochemical indicator for human telomeric DNA tetraplex.

A water-soluble perylene diimide derivative carrying two ferrocene moieties, FPD, was synthesized in two steps as an electrochemical indicator for human telomeric DNA tetraplex. Spectrophotometric binding studies of FPD with AG(3)(T(2)AG(3))(3) revealed its specific binding to tetraplex DNA in 10 mM MES and 1 mM EDTA (pH 6.25) containing 0.10 M KCl. Cyclic voltammograms of AAT CCG TCG AGC AGA G(T(2)AG(3))(4) in an electrolyte containing 20 mM FPN showed single oxidation and reduction peaks based on the ferrocene moieties. The peak current for the oxidation process increased linearly with the scan rate, suggesting the effective concentration of FPN on this electrode.

Synthesis of naphthalenediimide having two beta-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.

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.

Ferrocenylnaphthalene diimide-based electrochemical ribonuclease assay.

Messenger RNA (mRNA) poly(A)+RNA (from mouse kidney) was immobilized on a N-hydroxysuccinimide(NHS)-activated carboxylic acid modified electrode prepared by the treatment of a gold electrode with 3,3'-dithiodipropionic acid, followed by NHS and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). An electrochemical measurement using this mRNA electrode was carried out in an electrolyte containing ferrocenylnaphthalene diimide (1), and showed an electrochemical signal based on 1 concentrated on immobilized mRNA. After treating this electrode with water containing varied amounts of ribonuclease A (RNase A), the current peak based on 1 decreased with increasing in the amount of RNase A with a linear correlation in the range of 0.2-10 pg of RNase A.

Searching study for the suitable ferrocenylnaphthalene diimide derivative in the electrochemical telomerase assay.

Interaction of ferrocenylnaphthalene diimide 1 with the tetraplex oligonucleotide, AGGG(TTAGGG)3, which is a part of human telomere sequence, was studied in 0.1 M AcONa-AcOH (pH 5.5) containing 0.1 M NaCl coupled with 22-meric single and double stranded oligonucleotides using spectrophotometric titration experiment to search for the more suitable tetraplex DNA-binding ligand to achieve the electrochemical telomerase assay. CD spectra and polyacrylamide gel electrophoresis revealed that this tetraplex oligonucleotide kept to the single conformational structure of basket type G-quadruplex under these conditions. Scatchard analysis showed that 1 can bind to the tetraplex oligonucleotide with the binding constant of 10(5) M(-1) order, which was the highest value among the other oligonucleotides. Binding number of 1 for these oligonucleotides were ca. 2, 11, and 3 for tetraplex, double stranded, and single stranded oligonucleotides, respectively. These values were reasonable when considering with the binding mode of 1 as a threading intercalator.

Further stabilization of the double stranded DNA complex by naphthalene diimide derivative having 2,2-dipicolylamine-zinc complex.

Designed naphthalene diimide derivative having 2,2-dipicolylamine (Dpa), NDI-Dpa, could form a Zn(2+) complex and bind to calf thymus DNA with 10-times higher stabilization than that of NDI-Dpa. The obtained SDS-driven dissociation rate constant was 1.0 s(-1), which is smaller than that of NDI-Dpa. These results suggested that NDI-Dpa-Zn can bind to double stranded DNA through threading intercalation and its complex was stabilized by the interaction of Dpa-Zn parts of NDI-Dpa-Zn and phosphodiester parts of dsDNA.

Electrochemical RNase detection using ferrocenylnaphthalene diimide.

To develop a conventional RNase detecting system, an RNA electrode was constructed by the immobilization of poly(A)+RNA from mouse kidney on the glassy carbon electrode. Electrochemical measurement using this RNA electrode in the electrolyte containing ferrocenylnaphthalene diimide (1) was carried out and showed the electrochemical signal depending on the amount of the immobilized RNA. After this electrode was treated with water, the differential pulse voltammetric (DPV) measurement was subsequently conducted in the electrolyte containing 1. When RNase is contained in the water, the electrochemical signal decreased with an increase of the amount of RNase. This is derived from the decreasing amount of RNA on the electrode by RNase. In DPV measurement, the concentration of RNaseA was linear in the range of 10(-10) - 10(-8) M and the amount of RNase in the tap water could be estimated.

Preparation of carbodiimide-terminated dithiolane self-assembly monolayers as a new DNA-immobilization method.

A carbodiimide derivative having a dithiolane part at its terminus was designed and synthesized for use to construct carbodiimide-coated self-assembly monolayers (SAMs) on a gold surface with 6-mercaptohexanol (6MH). When treated with poly(dT), poly(dA), or poly(dA)poly(dT), only poly(dT) was immobilized on the surface of the SAMs through a specific reaction of the free imino moiety of thymine (T) with the carbodiimide moiety. The carbodiimide-covered SAM treated with probe DNA was tested in hybridization with sample DNA. Its hybridization efficiency was estimated by ferrocenylnaphthalene diimide (FND), described previously and the result revealed that the carbodiimide-covered SAM electrode can immobilize a DNA probe through the thymine moiety not involved in base pairing. The resulting electrode was capable of hybridizing with the target DNA, as proven by an increased current response of FND.

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.

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.

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.

Bis-naphthalene diimide exhibiting an effective bis-threading intercalating ability.

Bis-naphthalene diimide (1) was successfully synthesized from Fmoc-Lys(Boc)-OH and Fmoc-Lys(NDI)-OH on the automated peptide synthesizer. The absorption spectra of 1 suggested intramolecular stacking of the naphthalene diimide parts in buffer solution. Viscometric titration of a double stranded DNA (dsDNA) solution with 1 suggested that both of the naphthalene diimide parts of 1 are involved in threading intercalation. Kinetic studies suggested that 1 binds to dsDNA with a large affinity constant (K = 106 M(-1)) by "bis-threading intercalation" rather than by electrostatic binding between the cationic sites of lysine parts and phosphate anions of DNA.