Kinetics of Excimer Electrogenerated Chemiluminescence of Pyrene and 1-Pyrenebutyricacid 2-Ethylhexylester in Acetonitrile and an Ionic Liquid, Triethylpentylphosphonium Bis(trifluoromethanesulfonyl)imide.

We describe the kinetics of excimer electrogenerated chemiluminescence (ECL) of a liquid pyrene derivative, 1-pyrenebutyricacid 2-ethylhexylester (PLQ) dissolved in a molecular solvent, acetonitrile (MeCN), and an ionic liquid, triethylpentylphosphonium bis(trifluoromethanesulfonyl)imide ([P2225][TFSI]). Pyrene was also used for comparison. To discuss the kinetics of the excimer ECLs, the photophysical and electrochemical properties and electronic states of PLQ and pyrene were revealed. The photoluminescence (PL) spectra, rate constants for the radiative transitions, and redox potentials of PLQ and pyrene dissolved in MeCN and [P2225][TFSI] suggest that as a solvent, [P2225][TFSI] behaves more polar than MeCN. By analyzing the PL decay curves, the rate constants to form the excimer were determined to be on the order of 109 and 107 M-1 s-1 in MeCN and [P2225][TFSI], respectively, which were limited by the diffusion. For neat PLQ (1.6 M), a delay of 0.3-0.4 ns for the excimer emission compared to the monomer emission was observed. It is likely that the delay corresponds to the timescale for arranging the conformation to form the excimer. The ECL of PLQ was generated by applying a square wave voltage to produce the radical anion and cation, and on the ECL spectra, the excimer emission was more prevailed compared to the PL spectra. Kinetic analysis for the electron transfer reaction between the radical ions based on Marcus theory indicates that the electron transfer is limited by the diffusion of the radical ions. Moreover, the electron transfer distance (det) between the radical cation and anion to generate excited states was calculated with a framework of the theory. Kinetically, the electron transfer can take place at det < ∼11 Å in MeCN and det < ∼12 Å in [P2225][TFSI]. The density functional theory (DFT) and time-dependent DFT calculations show that the potential energy curve of the excimer against the distance between the pyrene rings reaches a minimum at 3.50 Å. This suggests that through the electron transfer, the process of the direct formation of the monomer S1 state followed by the excimer formation is more prevailed than that of the direct excimer formation.

Electrogenerated Chemiluminescence of Tris(dibenzoylmethane)phenanthroline Europium(III) as a Light Source: An Application for the Detection of PO43- Based on the Ion Associate Formation of Phosphomolybdic Acid and Malachite Green.

We demonstrate that electorogenerated chemiluminescence (ECL) of an organometallic Eu(III) complex, tris(dibenzoylmethane)phenanthroline europium(III), whose emission spectra is very sharp with the maximum wavelength of 612 nm, can be used as a light source for the detection of PO43- based on changing the absorbance of the ion associate of malachite green (MG+) and phosphomolybdic acid. The ECL was also applied to measure the absorbance of MG+. With the detection system we established, absorbance up to ∼1.6 with a change of ∼0.1 could be detected.

Efficient Electrogenerated Chemiluminescence of Pyrrolopyrrole Aza-BODIPYs in the Near-Infrared Region with Tripropylamine: Involving Formation of S2 and T2 States.

Efficient electrogenerated chemiluminescences (ECLs) of three pyrrolopyrrole aza-BODIPYs in the near-infrared region by using tripropylamine as a coreactant are reported. Kinetic analysis based on Marcus theory indicates the direct formation of S2 and T2 states through the electron transfer reaction, which affects the ECL efficiencies.

Totally synthetic microperoxidase-11.

A totally synthetic microperoxidase-11 (MP-11) is reported. Accordingly, the undecapeptide (VQKCAQCHTVE) was synthesized by solid-phase peptide synthesis followed by the thiol-ene click reaction with haemin for reconstitution. High-speed atomic force microscopy measurement conducted in water confirmed the protein reconstitution by visualizing the morphological differences as animated molecular images. The synthetic MP-11 showed a considerable magnitude of catalytic activity (27%) against the natural MP-11 in the oxidation of 3,3',5,5'-tetramethylbenzidine by hydrogen peroxide, whereas it showed very low (2.7%) activity of a synthetic variant with a point mutation (VQKCAQC M TVE, H8M). Slab waveguide spectroscopic measurements revealed that the ferrous/ferric redox reaction occurred by the direct electron transfer with specific spectral changes. Indeed, if hydrogen peroxide existed in the solution phase, the peroxidase-modified electrode showed catalytic current-voltage behaviour regardless of whether it was prepared using natural MP-11 or the synthetic MP-11. If a substrate recycling reaction was assumed, computer simulation well reproduced the experimental curves to give a global set of electrocatalytic reaction parameters. In any of the experiments, the synthetic MP-11 and natural MP-11 gave almost identical results. Our approach will be a convenient means of preparing MP-11, as well as its mutants, that does not rely on nature.

Monolithic Peptide-Nucleic Acid Hybrid Functioning as an Artificial Microperoxidase.

A new peptide nucleic acid (PNA) with an installed peroxidase function has been developed. Fmoc solid phase peptide synthesis prepared a PNA hybrid (VQKCAQCHTVE-(C2H4O)2CH2-[PNA(T)]6-G) that renders the microperoxidase backbone, followed by reconstitution with hemin. The resulting holocompound catalyzed the oxidation of 3,3',5,5'-tetramthylbenzidine by H2O2 to 50% that of natural microperoxidase-11, whereas the apo-form and hemin gave no responses. The peroxidase domain was found to be active toward direct electrochemistry and the PNA hybrid served for gene sensor; in the presence of the target DNA (5'-CATGTATAAAAAA-3'), an electrode-attached DNA probe (5'-TsTsTsTsTsTCTCATACATG-3') showed the ferric-to-ferrous quasi-reversible wave (-276 mV vs Ag/AgCl) through sandwich hybridization. Moreover, the hybridization product could accept H2O2 as an oxidant to enhance the reduction current, which occurred likely based on the iron(II)-center-recycling with specific rate constant of 0.19 s-1.

Determination of curcumin in biologically active supplements and food spices using a mesofluidic platform with fluorescence detection.

A mesofluidic platform (MP) with fluorescence detection based on a stepwise injection analysis (SWIA) was used for the determination of curcumin in biologically active supplements and food spices. The main units of the MP are a mixing chamber (MC) and an optical channel with a quartz capillary inside. The MC provides rapid and complete mixing solutions by gas bubbling. The proposed method is based on the new rapid and sensitive reaction of curcumin with a fluorescence reagent - 4-(2,3,3-trimethyl-3H-indolium-1-yl)butane-1-sulfonate (TIBS). The fluorescence intensity of TIBS is greatly quenched in the presence of curcumin in an alkaline medium. The linear range was from 1 to 10µM of curcumin, and the limit of detection, calculated as 3σ of a blank test (n=5), was found to be 0.3µM. The sample throughput was 24h(-1). The proposed method was successfully applied for the determination of curcumin in biologically active supplements and samples of food spices. The obtained data were in good agreement with those measured by a HPLC-UV method.

Development of a Portable Surface Plasmon Resonance Sensor with Multi-Sensing Points Based on the Linear CCD Sensor.

A portable-type surface plasmon resonance (SPR) sensor, composed from a new optical system for multi-sensing, has been developed to apply to environment analysis, clinical diagnosis etc., where many samples are desired to be analyzed at high throughput. The optical system of the sensor consists of a light-emitting diode, a pair of cylindrical lenses, a pair of collimator lenses, a correction lens, a prism, a polarizer and a linear CCD sensor with 2048 pixels. Reflected light from a sensor chip of the width of 6 mm at a certain incident angle was detected by ca. 618 pixels of the linear CCD sensor as an SPR sensor signal. An SPR sensor signal at a specified incident angle is controllable for optimization by adjusting the position of the CCD sensor. A sensor chip having a 30-stripe linear pattern (100 µm width/stripe) was prepared. The spatial resolution as well as the performance of the sensor were evaluated by using sucrose solutions. As a result, the acquisition of SPR sensor signals from 30 sensing points was successfully achieved with a spatial resolution of 100 µm (distance between 2 sensing points). A lower detection limit of ca. 3.2 - 5.5 × 10(-5) RIU with a standard deviation of ±4.5% was obtained by averaging the signals from 6 - 7 pixels of the CCD sensor per one sensing stripe.

Photophysical Properties and Efficient, Stable, Electrogenerated Chemiluminescence of Donor-Acceptor Molecules Exhibiting Thermal Spin Upconversion.

The photophysical properties and electrogenerated chemiluminescence (ECL) of three donor-acceptor molecules composed of dicyanobenzene and methyl-, tert-butyl-, and phenyl-substituted carbazolyl groups, 1,2,3,5-tetrakis(3,6-disubstituted-carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN-Me, 4CzIPN-tBu, and 4CzIPN-Ph, respectively) are described. These molecules show delayed fluorescence as a result of thermal spin upconversion from the lowest triplet state to the lowest singlet state at room temperature. The three molecules showed yellow to yellowish-red ECL. Remarkably, the ECL efficiencies of 4CzIPN-tBu in dichloromethane reached almost 40 %. Moreover, stable ECL was emitted from 4CzIPN-tBu and 4CzIPN-Ph. In case of 4CzIPN-Me, the ECL intensity decreased during voltage cycles because of polymerization. Quantum chemical calculations revealed that polymerization was inhibited by the steric hindrance of the bulky tert-butyl and phenyl groups on the carbazolyl moieties and lowered the spin density on the carbazolyl groups through electron conjugation for 4CzIPN-Ph.

Microfluidic White Organic Light-Emitting Diode Based on Integrated Patterns of Greenish-Blue and Yellow Solvent-Free Liquid Emitters.

We demonstrated a novel microfluidic white organic light-emitting diode (microfluidic WOLED) based on integrated sub-100-µm-wide microchannels. Single-µm-thick SU-8-based microchannels, which were sandwiched between indium tin oxide (ITO) anode and cathode pairs, were fabricated by photolithography and heterogeneous bonding technologies. 1-Pyrenebutyric acid 2-ethylhexyl ester (PLQ) was used as a solvent-free greenish-blue liquid emitter, while 2,8-di-tert-butyl-5,11-bis(4-tert-butylphenyl)-6,12-diphenyltetracene (TBRb)-doped PLQ was applied as a yellow liquid emitter. In order to form the liquid white light-emitting layer, the greenish-blue and yellow liquid emitters were alternately injected into the integrated microchannels. The fabricated electro-microfluidic device successfully exhibited white electroluminescence (EL) emission via simultaneous greenish-blue and yellow emissions under an applied voltage of 100 V. A white emission with Commission Internationale de l'Declairage (CIE) color coordinates of (0.40, 0.42) was also obtained; the emission corresponds to warm-white light. The proposed device has potential applications in subpixels of liquid-based microdisplays and for lighting.

A Miniaturized Stepwise Injection Spectrophotometric Analyzer.

A novel micro-stepwise injection analyzer (µSWIA) has been developed for the automation and miniaturization of spectrophotometric analysis. The main unit of this device is a mixing chamber (MC) connected to the atmosphere. This part of the µSWIA provides rapid and effective homogenization of the reaction mixture components and completion of the reaction by means of gas bubbling. The µSWIA contained a rectangular labyrinth channel designed in way allowing one to eliminate bubbles by moving a solution from the MC to an optical channel. The light-emitting diode (LED) was used as a light emitter and the analytical signal was measured by a portable spectrophotometer. Fluid movement was attained via the use of a computer-controlled syringe pump. The µSWIA was successfully used for the spectrophotometric determination of cysteine in biologically active supplements and fodder by using 18-molybdo-2-phosphate heteropoly anion (18-MPA) as the reagent.

Fluorometric flow-immunoassay for alkylphenol polyethoxylates on a microchip containing a fluorescence detector comprised of an organic light emitting diode and an organic photodiode.

A compact fluorescence detector was constructed on a microchip from an organic light emitting diode (OLED) as the light source and an organic photodiode (OPD) as the photo-detector and was used in an immunoassay for alkylphenol polyethoxylates (APE). The OLED based on a terbium complex emitted a sharp light at the main wavelength of 546 nm with a full width at half maximum of 9 nm. The incident photo-to-current conversion efficiency (IPCE) of the OPD fabricated with Fullerene 70 (C70) and tris[4-(5-phenylthiopen-2-yl)phenyl]-amine (TPTPA) was approximately 44% for light at a wavelength of 586 nm. The performance of the fluorescence detector was evaluated for the determination of resorufin (λ(em)=586 nm) and the photocurrent of the OPD due to the fluorescence of resorufin was proportional to the concentration of resorufin in the range from 0 to 18 µM with a detection limit (S/N=3) of 0.6 µM. The fluorescence detector was successfully utilized in a competitive enzyme-linked immunosorbent assay for APE, where an anti-APE antibody was immobilized on the surface of the channel of the Polydimethylsiloxane (PDMS) microchip or on the surface of magnetic microbeads. After an immunoreaction with a sample solution of APE containing a horse radish peroxidase (HRP)-labeled APE, the fluorescence of resorufin generated just after introduction of a mixed solution of Amplex Red and H2O2 was measured using the fluorescence detector. The calibration curve for the photocurrent signals of the OPD due to the fluorescence of resorufin against the logarithmic concentration of APE was sigmoidal in shape. The detection limits defined as IC80 were ca. 1 ppb and ca. 2 ppb, respectively, for the methods using the anti-APE antibody immobilized on the surface of the microchannel and in the case where the antibody was immobilized on the surface of magnetic microbeads.

Automated chemiluminescence immunoassay for a nonionic surfactant using a recycled spinning-pausing controlled washing procedure on a compact disc-type microfluidic platform.

A fully automated and integrated chemiluminescence immunoassay, carried out on a compact disc (CD)-type microfluidic platform, for the detection of alkylphenol polyethoxylates (APnEOs) is described. The pattern of the CD-type microchip was designed so as to permit the sequential solution delivery of the sample solution, the washing solution and the luminol solution, which are required in the chemiluminescence immunoassay process, along with a designed rotation program for spinning the CD-type microchip. The procedure for flowing the washing solution, the volume of which was limited on the CD-type microchip, was optimized by using a recycled spinning-pausing rotation program to overcome the non-specific adsorption of the horseradish peroxidase labeled APnEOs at the detection area. The detection limit of the immunoassay is about 10 ppb.

Photometric flow injection determination of phosphate on a PDMS microchip using an optical detection system assembled with an organic light emitting diode and an organic photodiode.

A compact photometric detector was constructed from an organic light emitting diode (OLED) based on a europium complex, europium(diben-zoylmethanato)3(bathophenanthroline) (Eu(DBM)3bath), as the light source and an organic photodiode (OPD) fabricated from a hetero-junction of two layers of copper phthalocyanine (CuPc)/fullerene (C60) as the photo-detector on a microchip prepared from poly(dimethylsiloxan) (PDMS) and was applied to the determination of phosphate. The OLED and the OPD were fabricated by a vapor deposition method on an indium tin oxide (ITO) coated glass substrate with the following layered structure; Glass (0.7 mm)/ITO (110 nm)/4,4'-bis[N-(1-naphthyl)-N-phenyl amino]-biphenyl (α-NPD) (30 nm)/4,4'-di(N-carbazolyl)biphenyl (CBP): Eu(3+) (8 wt%, 30 nm)/bathocuproine (BCP) (30 nm)/aluminum tris(8-hydroxyquinoline) (Alq3) (25 nm)/magnesium and silver (MgAg) (100 nm)/Ag (10nm) and Glass (0.7 mm)/ITO (110 nm)/CuPc (35 nm)/C60 (50 nm)/BCP (10 nm)/Ag (50 nm), respectively. The OLED based on the europium complex emitted a sharp light at the wavelength of 612 nm with a full width at half maximum (FWHM) of 8 nm. The performance of the photometric detector assembled was evaluated based on measurements of the absorbance of different concentrations of malachite green (MG) solutions for a batch system with 1cm long path length. The molar absorptive coefficient of the MG solution, calculated from the photocurrent of the OPD, was in good agreement with the value reported in the literature. A microchip with two inlets and one outlet U-shaped channel was prepared by a conventional photolithograph method. The OLED and the OPD were configured so as to face each other through the PDMS microchip in parallel in order to align the light axis of the OLED and the OPD with the flow cell (optical path length of 5mm), which was located at the end of outlet. For the determination of phosphate, an ion-association reaction between MG and a molybdenum-phosphate complex was utilized and a good linear relationship between the concentration and absorbance was observed in the concentration range 0-0.2 ppm, with a detection limit (S/N=3) of 0.02 ppm. The assembled photometric detector was also applied to the determination of phosphate by the flow injection of river water samples using the reagent solution containing MG and molybdenum ammonium in sulfuric acid. A good recovery (97-99%) for the river water samples, which had been spiked with the standard 0.08 ppm, with an RSD of ca 5% (n=5) was obtained using the constructed system.

Optical characterization of the antigen-antibody thin layer using the whispering gallery mode.

We immobilized an antibody (anti-ß-Galactosidase) on a polystyrene microsphere by using a covalent bond, and observed the resonance peaks in the scattered light intensity spectra related to the whispering gallery mode (WGM) excitation of the microsphere. The amount and the optical parameters, i.e., thickness and refractive index, of anti-ß-Galactosidase on the sphere surface were evaluated based on an absorbance measurement and a resonance peak shift measurement, respectively. Moreover, we measured the variation of the WGM spectra depending on the concentration of the enzyme solution (ß-Galactosidase), which allowed us to optically evaluate the thickness and the refractive index of the antigen-antibody layer from the shift of the WGM spectra peak.

Electrogenerated chemiluminescence of donor-acceptor molecules with thermally activated delayed fluorescence.

The electrochemistry and electrogenerated chemiluminescence (ECL) of four kinds of electron donor-acceptor molecules exhibiting thermally activated delayed fluorescence (TADF) is presented. TADF molecules can harvest light energy from the lowest triplet state by spin up-conversion to the lowest singlet state because of small energy gap between these states. Intense green to red ECL is emitted from the TADF molecules by applying a square-wave voltage. Remarkably, it is shown that the efficiency of ECL from one of the TADF molecule could reach about 50%, which is comparable to its photoluminescence quantum yield.

Sequential injection analysis of thiocyanate ions using a microfluidic polymer chip with an embedded ion-selective electrode.

A sequential injection analysis (SIA) system for the determination of SCN(-) ion using a microfluidic polymer chip with an embedded SCN(-) ion-selective electrode (SCN(-)-ISE) as a detector was developed. Under the optimal flow conditions of the SIA system, the SCN(-)-ISE showed a good linear relationship between peak heights and logarithmic concentrations of SCN(-) ion with a Nernstian slope of 59.4 ± 3.8 mV decade(-1) in a concentration range from 1.0 × 10(-5) to 1.0 × 10(-1) mol dm(-3). Sample throughput of the present system for the determination of SCN(-) ion was approximately 12 samples h(-1). The proposed SIA system was applied to the determination of the level of SCN(-) ion in human saliva samples.

An organic thin film photodiode as a portable photodetector for the detection of alkylphenol polyethoxylates by a flow fluorescence-immunoassay on magnetic microbeads in a microchannel.

An organic thin film photodiode (OPD) was successfully employed as a portable photodetector in a competitive enzyme-linked immunosorbent assay (ELISA) of a class of nonionic surfactants, namely alkylphenol polyethoxylates (APnEOs) which are an environmental pollutant. Microbeads that were chemically immobilized with an anti-APnEOs antibody were used in the assay. The OPD consisted of a layer of copper phthalocyanine (CuPc), C60 and a second layer of bathocuproine (BCP) with a bulk heterojunction composed of CuPc and C60 prepared by a vapor deposition method on an indium-tin oxide coated glass substrate. The OPD showed an incident photon-current efficiency (IPCE) of approximately 19% for light at a wavelength of 585 nm. This relatively high IPCE at 585 nm makes it suitable for detecting the fluorescence of resorufin (λem=585 nm), the product of the competitive ELISA, produced through the enzymatic reaction of Amplex Red with horseradish peroxidase (HRP) and H2O2. A fluorometric detector was assembled on a microchip by combining the fabricated OPD and a commercial LED as a photodetector and a light source, respectively. The photocurrent of the OPD due to the fluorescence of resorufin was proportional to the concentration of resorufin in the concentration range from 0 to 8 µM. When the fabricated OPD was used as a portable photodetector, the competitive ELISA of APnEOs using HRP labeled APnEOs (HRP-APnEOs) was performed on magnetic microbeads on which surface an anti-APnEOs antibody had been immobilized. A typical sigmoidal calibration curve was obtained and the data were in good agreement with a numerical simulation, where the photocurrent of the OPD was plotted against the concentration of APnEOs, determined via the competitive ELISA. The detection limit of the immunoassay for APnEOs was approximately 2 and 4 ppb in batch and flow system, respectively.

Solvent effect on thermally activated delayed fluorescence by 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene.

Thermally activated delayed fluorescence (TADF) is fluorescence arising from a reverse intersystem crossing (RISC) from the lowest triplet (T1) to the singlet excited state (S1), where these states are separated by a small energy gap (ΔEst), followed by a radiative transition to the ground state (S0). Rate constants relating TADF processes in 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN) were determined at four different solvent polarities (toluene, dichloromethane, ethanol, and acetonitrile). We revealed that the rate constant of RISC, kRISC, which is the most important factor for TADF, was significantly enhanced by a reduced ΔEst in more polar solvents. The smaller ΔEst was mainly attributable to a stabilization of the S1 state. This stabilization also induced a Stokes shift in fluorescence through a relatively large change of the dipole moment between S1 and S0 states (17 D). Despite of this factor, we observed a negative correlation between ΔEst and efficiency of the delayed fluorescence (φd). This was ascribed to a lower intersystem crossing rate, kISC, and increased nonradiative decay from S1, k(s)nrs, in polar solvents.

Development of a novel two dimensional surface plasmon resonance sensor using multiplied beam splitting optics.

A novel two dimensional surface plasmon resonance (SPR) sensor system with a multi-point sensing region is described. The use of multiplied beam splitting optics, as a core technology, permitted multi-point sensing to be achieved. This system was capable of simultaneously measuring nine sensing points. Calibration curves for sucrose obtained on nine sensing points were linear in the range of 0-10% with a correlation factor of 0.996-0.998 with a relative standard deviation of 0.090-4.0%. The detection limits defined as S/N = 3 were 1.98 × 10(-6) - 3.91 × 10(-5) RIU. This sensitivity is comparable to that of conventional SPR sensors.

A pivot-hinge-style DNA immobilization method with adaptable surface concentration based on oligodeoxynucleotide-phosphorothioate chemisorption on gold surfaces.

The chemisorption of oligodeoxynucleotide phosphorothioate (s-oligo) is reported. A series of s-oligo DNAs was designed for use as capture probe DNA molecules. The s-oligo DNAs consist of the K-ras gene (5'-GGA GCT GGT GGC-3') and a dodecamer deoxyriboadenosine, both of which lie on either side of an s-oligo DNA sequence. By primarily focusing on the capture probe DNA having five-successive s-oligo sequences, e37, the immobilization chemistry of e37 was examined; atomic force microscopy achieved the direct visualization of individual molecules on Au(111) substrates, while a series of surface analyses, including IR, ellipsometry, and microgravimetry, showed that the s-oligo functional groups played a pivotal role in the surface-adlayer through the gold-thiol interaction. Interestingly, the amount of immobilization showed a definite relationship with the number of s-oligo linkages introduced, which should be important to regulate the concentration of the capture probe DNA molecules on the surface. Some preliminary studies using ferrocene-modified complementary sequences indicated that electrochemical labeling and readouts were possible.