Ik-Soo Shin

Fluorescence Turn-On Sensor for Cyanide Based on a Cobalt(II)−Coumarinylsalen Complex

A Co(II)-salen based fluorescent sensor (1.Co) that can selectively recognize cyanide anions in 1:2 binding stoichiometry over other anions has been developed. 1.Co displayed fluorescence enhancement upon the addition of cyanide owing to the interruption of photoinduced electron transfer from the coumarin fluorophore to the cobalt(II) ion. A general regression method was developed to calculate the binding constants in the 1:2 binding system, through which the 1:2 binding between 1.Co and cyanide anions was estimated to be in the range of micromolar dissociation constants.

Detection of Kinase Activity Using Versatile Fluorescence Quencher Probes

Protein phosphorylation is the most universal form of posttranslational modification of cell-signal transduction in living organisms. The human kinome comprises 518 protein kinases that control protein phosphorylation; irregular control of protein phosphorylation is a major cause of diseases such as cancer. Therefore, accurate probing of the kinase activity of a target protein is crucial for cancer diagnosis and high-throughput screening of anticancer drugs. 3] For the high-throughput analysis of kinase activity, several research groups have developed various types of protein or peptide chips using radioactive labeling with [Pg]-adenosine 5’-triphosphate (ATP) or using antibody hybridization. However, a crucial problem involved in the use of these onchip detection methods is that some kinases show decreased activities on the surfaces of chips because of reduced enzyme accessibility to the substrate. In recent years, several pharmaceutical and biotechnology companies have developed homogeneous kinase assay systems based on fluorescence polarization (FP) for developing anticancer drugs. These platforms (kinase assay systems) utilize peptide substrates with an N-terminal fluorophore and phospho-specific antibodies or phosphopeptide (or phosphoprotein)-binding nanoparticles (IMAP). However, FPbased detection has been reported to be very sensitive to fluorescence interference, and it is liable to produce false positives when used to screen a large number of compounds. Furthermore, there are no reports on the real-time monitoring of kinase activity in cell lysates through FP-based kinase detection; this is because many cellular components can bind to the fluorescent peptides and produce false positives for FP. Recently, peptideor protein-linked synthetic fluorescent probes that are sensitive to certain protein kinases have been reported by the research groups of Lawrence, Imperiali, Sames, and Hamachi. Ting, Tsien, and co-workers used fluorescent proteins to develop an in vivo probe system to detect kinase. These synthetic probes enabled real-time fluorescence monitoring of the specific activity of kinases in cellular lysates, and exhibited immense potential for use in the development of kinase activity inhibitors for certain kinases. However, it is still difficult to predict and determine the optimal sites for attaching fluorophores near the phosphorylated sites on the substrate peptides or proteins; the attachment of these fluorophores is necessary to induce significant changes in the fluorescence signal after phosphorylation of the substrate peptides or proteins by a specific kinase. Therefore, a general strategy for developing a synthetic fluorescent kinase probe is desired. We designed chemosensors Dab-DPA and PTZ-DPA (Scheme 1) to develop a simple but powerful kinase assay tool based on fluorescence intensity changes (ON/OFF). Using these chemosensors, we show for the first time the diagnosis of chronic myelogenous leukemia (CML) through real-time fluorescence monitoring of Abelson (Abl) tyrosine kinase activity and the development of a fluorescence-based homogeneous kinase assay system on a microfluidic chip. As shown in Scheme 1, Dab-DPA consists of a bis(Zndipicolylamine) complex and a dabcyl (Dab) fluorescence quencher, and PTZ-DPA consists of the dipicolylamine complex and a phenothiazine (PTZ) fluorescence quencher. Dab and PTZ quench fluorescence by F rster resonance energy transfer (FRET) and photoinduced electron transfer (PET), 11g] respectively. The bis(Zn-dipicolylamine) complex is a well-known synthetic receptor that strongly and selectively binds to phosphate in aqueous solution. DabDPA and PTZ-DPA are synthesized in a few steps (see the Supporting Information). PTZ is a good fluorescence quencher but there are very few reports on its use as such, except for the isoalloxazine ring of flavins. 11g] To prove that PTZ can be used as a general fluorescence quencher for other fluorophores, such as carboxyfluorescein (FAM) or tetramethyrhodamine (TMR), we performed electrochemical analyses of PTZ, FAM, and TMR. Figure 1a shows the cyclic voltammograms of 1 mm PTZ, TMR, and FAM; the Pt-disk working electrode is immersed in acetonitrile with 0.1m tetrabutylammonium hexafluorophosphate (TBAPF6) as supporting electrolyte. The observed waves are assigned to the oxidation of PTZ and the fluorophores (TMR and FAM). PTZ undergoes nearly Nernstian oxidation at E1/2,ox = 0.63 V with a peak separation [*] Dr. H.-W. Rhee, S. H. Lee, Dr. I.-S. Shin, S. J. Choi, Prof. Dr. T. H. Park, Prof. Dr. J.-I. Hong Department of Chemistry School of Chemical & Biological Engineering Seoul National University, Seoul 151-747 (Korea) Fax: (+ 82)2-889-1568 E-mail: thpark@snu.ac.kr jihong@snu.ac.kr

Efficient Fluorescence “Turn-On” Sensing of Dissolved Oxygen by Electrochemical Switching

We report on a novel method for sensing oxygen that is based on the use of a perylene diimide dye (1) which is electrochemically reduced to its nonfluorescent dianion form (1(2-)). In the presence of oxygen, the dianion is oxidized to its initial form via an electron-transfer reaction with oxygen upon which fluorescence is recovered. As a result, the fluorescence intensity of the dianion solution increases upon the addition of oxygen gas. Results demonstrate that high sensitivity is obtained, and the emission intensity shows a linear correlation with oxygen content (0.0-4.0% v/v) at ambient barometric pressure. In addition, using electrochemical reduction, oxygen determination becomes regenerative, and no significant degradation is observed over several turnovers. The limit of detection is 0.4% oxygen in argon gas.

Analytical detection of biological thiols in a microchip capillary channel.

Sulfur-containing amino acids, such as cysteine and homocysteine play crucial roles in biological systems for the diagnosis of medical states. In this regard, this paper deals with separation, aliquot and detection of amino thiols on a microchip capillary electrophoresis with electrochemical detection in an inverted double Y-shaped microchannel. Unlike the conventional capillary electrophoresis, the modified microchannel design helps in storing the separated thiols in different reservoirs for further analysis, if required; and also eliminates the need of electrodes regeneration. The device was fabricated using conventional photolithographic technique which consisted of gold microelectrodes on a soda lime glass wafer and microchannels in PDMS mold. Multiple detections were performed using in-house fabricated dual potentiostat. Based on amperometric detection, cysteine and homocysteine were analyzed in 105 s and 120 s, respectively after diverting in branched channels. Repeated experiments proved the good reproducibility of the device. The device produced a linear response for both cysteine and homocysteine in electrochemical analysis. To prove the practicality of device, we also analyzed cysteine and homocysteine in real blood samples without any pre-treatment. Upon calculation, the device showed a very low limit of detection of 0.05 μM. The modified microchip design shall find a broad range of analytical applications involving assays of thiols and other biological compounds.

Homogeneous Electrochemical Assay for Protein Kinase Activity

Herein, we report a homogeneous assay for protein kinase activity using an electrochemistry-based probe. The approach involves a peptide substrate conjugated with a redox tag and the phosphate-specific receptor immobilized on an electrode surface. The peptide substrate phosphorylated by a protein kinase binds to the receptor site of the probe, which results in a redox current under voltammetric measurement. Our method was successfully applied even in the presence of citrated human blood and modified to enable a single-use, chip-based electrochemical assay for kinase activity.

Evaluation of electrogenerated chemiluminescence from a neutral Ir(III) complex for quantitative analysis in flowing streams

Though recently Ir(III) complexes have attracted much interest in electrochemiluminescent (ECL) analysis due to their high emission in various wavelengths, there were a few studies reported on its analytical applications. In this study, we evaluate the ECL from (pq)(2)Ir(acac) (pq = 2-phenylquinolate, acac = acetylacetonate) for the use in flow injection analysis. An aqueous solution of the analyte and (pq)(2)Ir(acac) passes through the reaction/observation cell, and then ECL reaction is generated by electrochemical initiation on the analyte and (pq)(2)Ir(acac). Tri-n-propylamine (TPrA) is used as a representative analyte for evaluation. Additionally, a comparison is made of the relative ECL intensities obtained for a variety of analytes including oxalate, amino acids, aliphatic amines, and NADH. The (pq)(2)Ir(acac) produces efficient ECL upon TPrA exhibiting the limit of detection of 5 nM with a linear range of 3 orders of magnitude in concentration whereas 20 nM is observed in the conventional Ru(bpy)(3)(2+) system. It shows particular sensitivity advantages for oxalate, proline, and tartaric acid. The ECL generation upon various analytes proposes direct applicability of (pq)(2)Ir(acac) as a post-column detection tool.

Microfluidic bead-based sensing platform for monitoring kinase activity

Protein kinases control cellular functions by regulating protein phosphorylation. Monitoring protein kinase activity is essential for medical diagnosis and drug screening. Here, we present a novel microfluidic device for performing simple and versatile protein kinase assays, which utilizes a microbead-based chemosensor. An automatic mix-and-measure technique was achieved using integrated pneumatic valves. After mixing each reagent for the kinase assay, the mixture was transferred to the sensing chamber. Then, phosphorylated and fluorescence-labeled peptides were captured and detected by the chemosensor. A fluorescence signal was observed depending on the presence of the kinase. Furthermore, activities of various kinases in the cell lysate and the inhibitory effect of specific chemicals on the kinases were monitored. These results indicate that chemosensor-based microfluidic chips can be developed as a versatile kinase assay system.

Nonvolatile floating gate organic memory device based on pentacene/CdSe quantum dot heterojuction

An organic floating-gate memory device using CdSe quantum dots (QDs) as a charge-trapping element was fabricated. CdSe QDs were localized beneath a pentacene without any tunneling insulator, and the QD layer played a role as hole-trapping sites. The band bending formed at the junction between pentacene and QD layers inhibited back-injection of holes trapped in CdSe into pentacene, which appeared as a hysteretic capacitance-voltage response during the operation of the device. Nearly, 60% of trapped charge was sustained even after 104 s in programmed state, and this long retention time can be potentially useful in practical applications of non-volatile memory.

Efficient blue phosphorescent host through nonbonded conformational locking interactions

We developed a new host material TCTEB with a conformationally homogeneous, sterically bulky structure due to nonbonded interactions between adjacent methylene groups. The host presented good thermal stability (Tg = 122 °C and Td = 321 °C) and high triplet energy (3.0 eV). The maximum quantum efficiency, current efficiency and power efficiency of the OLED device using the TCTEB host were 8.5%, 13.7 cd A−1, and 11.3 lm W−1, respectively, when doped with 8 wt% FIrpic.

Electrochemiluminescent chemodosimeter based on iridium(III) complex for point-of-care detection of homocysteine levels

Elevated levels of plasma homocysteine (Hcy) are an independent risk factor for cardiovascular disease. Although a routine, rapid, and simple determination of Hcy levels is highly desired, the existing methods are practically limited because of complicated sample preparation and bulky instrumentation. Herein, we report a chemodosimetric approach for one-step analysis of Hcy levels based on the electrochemiluminescence (ECL). A rationally designed cyclometalated iridium(III) complex possessing a phenylisoquinoline main ligand underwent a selective ring-formation reaction with Hcy to generate a binding adduct, which enabled producing highly luminescent excited states, and yielded strong ECL signals on the surface of electrode without any use of enzymes or antibodies. The level of Hcy was successfully monitored by the ECL increment with a linear correlation between 0 and 40µM in 99.9% aqueous media. The approach required neither sample preparation nor bulky instrument, suggesting the point-of-care testing of Hcy levels, and is potentially useful for routine, cost-effective, and precautionary diagnosis of various cardiovascular diseases.