Hizuru Nakajima

Antimony-film electrode for the determination of trace metals by sequential-injection analysis/anodic stripping voltammetry

The possibility of applying antimony-film modified glassy carbon electrode in sequential-injection analysis (SIA) was investigated with the objective of determining Pb(II) and Cd(II) by anodic stripping voltammetry (ASV). The conditions of antimony-film deposition concerning composition of the plating/carrier solutions, concentrations of Sb(III) and hydrochloric acid, effects of different supporting electrolyte salts, and plating potential were optimized. It was found that the antimony-film deposition on glassy carbon substrate in a sample solution consisting of 750 microg L(-1) Sb(III), 0.5 mol L(-1) HCl at -1.5 V (vs. Ag/AgCl/3 mol L(-1) KCl) yielded a modified electrode suitable for the determination of Pb(II) and Cd(II) at the microg L(-1) level. The reproducibility of the analytical signals was characterized by a relative standard deviation lower than 2.8%, and the calculated values of detection limits were 1.2 microg L(-1) for Pb(II) and 1.4 microg L(-1) for Cd(II). The presence of KSCN in the sample solution offers the possibility of detecting ions with more negative oxidation potentials like Zn(II), Mn(II) or Cr(III). The developed SIA-ASV procedure was compared with the commonly used batch method, and its applicability was tested on a spiked tap water sample.

Inkjet Nanoinjection for High-Thoughput Chemiluminescence Immunoassay on Multicapillary Glass Plate

We report a novel chemiluminescence diagnosis system for high-throughput human IgA detection by inkjet nanoinjection on a multicapillary glass plate. As proof-of-concept, microhole-based polydimethylsiloxane (PDMS) sheets were aligned on a multicapillary glass plate to form a microwell array as microreactors for enzyme-linked immunosorbent assay (ELISA). The multicapillary glass plate was utilized as a switch that controlled the holding/passing of the solution. Further, anti-IgA-labeled polystyrene (PS) microbeads was assembled into the microwell array, and an inkjet nanoinjection was specially used to distribute the sample and reagent solution for chemiluminescence ELISA, enabling high-throughput detection of human IgA. As a result, the performance of human IgA tests revealed a wider range for the calibration curve and a lower limit of detection (LOD) of 0.1 ng mL(-1) than the ELISA by a standard 96-well plate. The analysis time and reagent consumption were significantly decreased. The IgA concentrations in saliva samples were determined after 10000-fold dilution by the developed ELISA system showing comparable results by conventional immune assay with 96-wells. Thus, we believe that the inkjet nanoinjection for high-throughput chemiluminescence immunoassay on a multicapillary glass plate will be promising in disease diagnosis.

Perylenebisimide-linked nitroxide for the detection of hydroxyl radicals

A new type of fluorophore-linked nitroxide for the detection of hydroxyl radical (OH) was synthesized using perylene-3,4,9,10-tetracarboxyl bisimide and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). This new fluorescent probe, indicated here as Spy-OH (swallow-tailed perylene derivative for detecting OH) has its natural fluorescence effectively reduced due to the quenching caused by the presence of the nitroxide radical in its structure. In the presence of OH, in media containing DMSO, a conspicuous increase in the fluorescence intensity was observed. No significant change in the fluorescence intensity was observed upon the addition of other several reactive oxygen species (ROS) except for OH. Spy-OH maxima excitation (lambda(exc)) and emission (lambda(em)) wavelengths lie in the visible region (520/530 nm), therefore, this fluorescent probe is expected to be useful for a simple and selective detection of OH, especially for biosamples.

Development of an automatic multi-channel ink-jet ejection chemiluminescence system and its application to the determination of horseradish peroxidase.

In this work, an automatic multi-channel ink-jet for chemiluminescence (CL) analysis was developed. The four-channel ink-jet device was controlled by a home-made circuit. Differing from the classic flow injection CL, the whole procedure for CL analysis was automatically completed on a hydrophobic glass side. CL reaction of luminal and hydrogen peroxide for the determination of horseradish peroxidase (HRP) was selected as an application to automatic CL analysis platform. All solutions delivered by different channels were precisely ejected to the same position of the glass slide for the CL analysis. The consumption of reaction solution was reduced to nanoliter level. The whole CL analysis could be completed in less than 4min, which was benefited from the prompt solution mixing in small size of droplet. The CL intensity increased linearly with HRP concentration in the range from 0.01 to 0.5μgmL(-1). The limit of detection (LOD) (S/N=3) was 0.005μgmL(-1). Finally, the automatic CL system could also be used for the detection of HRP in HRP-protein conjugates, which showed its practical application in immunoassay.

Accurate and highly reproducible picoliter injection system for capillary electrophoresis.

A novel, highly accurate sample injection system for capillary electrophoresis (CE) was developed based on an inkjet microchip capable of reproducing exact introduction volumes at the picoliter level. The difficulty in analyte discrimination using electrokinetic injection was also overcome using this injection method. The injection system consisted of an XY stage, an inkjet droplet ejection microchip, and a reservoir with a plug-in septum. To evaluate the precision of the system, a mixture of NBD-labeled amino acids consisting of Gly, L-Phe, L-Asp, and L-Ser was separated, and the performance was compared with that of traditional hydrodynamic and electrokinetic injection methods. The results demonstrated that the introduced volume highly relied on the number of droplets with low relative standard derivation (RSD) and good linear correction coefficient in the proposed injection method. In addition, a urine sample was analyzed via CE coupled with the inkjet injection system for the detection of the amino acid taurine. The concentration of urinary taurine was determined to be 2.42 ± 0.08 μM (confidence level, 95%; RSD, 1.05%; n = 4) with a recovery of 98.92-109.54% (n = 3). These results demonstrate the inkjet injection system we developed has the potential to revolutionize capillary electrophoretic separation in practical and commercial applications that require an automated accurate injection system.

Performance of an organic photodiode as an optical detector and its application to fluorometric flow-immunoassay for IgA.

The performance of an organic thin film photodiode (OPD), fabricated from a hetero-junction comprised of two layers of C(60) and a phthalocyanine-Cu(II) complex was evaluated by detecting the chemiluminescence generated from the reaction of luminol with horseradish peroxidase in the presence of H(2)O(2), and the fluorescence from resorufin, as an optical detector. The photocurrent of the OPD was linear with respect to the power of light from a commercial LED. The sensitivity of the OPD was sufficient for detecting chemiluminescence with a power 0.1μW/cm(2). The OPD was successfully used in a flow-immunoassay for IgA, a marker of human stress, in which a sandwich immunoassay was carried out on the microchip and the fluorescence from resorufin, produced by the enzymatic reaction, was detected. The detection limits for resorufin and IgA were 5.0μM and 16ng/mL, respectively. The photosensitivity of the OPD remained relatively constant for a minimum of one year.

A flow-based enzyme-linked immunosorbent assay on a polydimethylsiloxane microchip for the rapid determination of immunoglobulin A.

A flow-based enzyme-linked immunosorbent assay (ELISA) on a polydimethylsiloxane (PDMS) microchip has been developed for the rapid determination of immunoglobulin A (IgA). The analytical principle of this integrated method is the same as the conventional sandwich-type ELISA. A primary antibody (anti-IgA) was adsorbed on the surface of a PDMS microchannel, and then an antigen (IgA) and a secondary antibody (anti-IgA HRP labeled) were reacted successively. The resulting antigen-antibody complex, fixed on the surface of the microchannel, was detected using Amplex((R)) Red and a fluorescent imaging system. The calibration curve of the IgA standard solution was linear in the range of 0-50ng/mL at the flow rate of 10muL/min. This flow rate corresponds to the reaction time of 4.8s. Compared to the conventional assay on a 96-well microtiter plate, the present assay on the microchip dramatically shortened the reaction time necessary for the enzyme-substrate reaction from 30min to 4.8s, i.e., to 1/375. The amounts of the reagent and sample were also reduced to 1/100 compared to the 96-well microtiter plate.

A palm-sized surface plasmon resonance sensor with microchip flow cell

A small-sized surface plasmon resonance (SPR) sensor with a microchip flow cell has been developed for the purpose of enhancing the sensitivity of the SPR detector for low molecular weight compounds. This portable differential SPR detector consisted of an LED, two cylindrical lenses, a round prism, a divided mirror, a CCD, electronics, and a polydimethylsiloxane/gold microchip with two flow paths (10mm long, 1mm wide, 20-100mum deep). 3-Mercaptopropyltrimethoxysilane was used for sealing the microchip. The performance of the on-site orientated SPR detector was estimated using sucrose and IgA. A drastic change in the SPR intensity appeared. The depth of the flow cell was in inverse proportion to the SPR intensity. Compared to a conventional flow cell having the size of 10mm (L)x1mm (W)x1mm (D), its sensitivity to 10% sucrose and 0.9nM IgA increased about 11 and 39 times, respectively. This phenomenon seemed to be due to the increase in the substance on the SPR sensor based on its size effect. These results showed that the application of the microchip sensor for SPR measurement has the possibility for improvement of the SPR intensity for low molecular substances.

A piezoelectric drop-on-demand generator for accurate samples in capillary electrophoresis

In this work, we propose a piezoelectric droplet generator for injection of well-defined amounts of sample in capillary electrophoresis. We demonstrate stable, precise and drop-on-demand droplet formation for various solutions, with precise control of waveform driving piezoelectric crystal inside the ink-jet head. By tuning the waveform, we can also manipulate the droplet size and delivery frequency. This injector was used in sampling for capillary electrophoresis. As a state-of-the-art application, the analysis of theobromine, caffeine and theophiline using micellar electrokinetic chromatography was developed. The volume of sample (single droplet) analyzed in this experiment was 179 pL (RSD=1.2%, n=10). The detection limits for caffeine, theobromine, and theophiline are 0.02, 0.08 and 0.06 mM L(-1), respectively. Compared with conventional methods, the combination of picoliter droplet dispenser with capillary electrophoresis allows precise and accurate sampling, as well as for reduced sample consumption, which will prove to be an efficient tool in quantitative separation and analysis.

A surface plasmon resonance sensor on a compact disk-type microfluidic device

A surface plasmon resonance (SPR) sensor on a compact disk (CD)-type microfluidic device was developed to miniaturize the elements of a complete analytical system, pump and valves. The CD-type microfluidic device was fabricated by attaching a polydimethylsiloxane disk plate that contained microchannels and reservoirs to a flat polycarbonate disk plate that contained grating films with a thin layer of Au. The optical system of the SPR sensor and the theory for its operation are based on the principle of a grating coupled-type SPR. The sample and reagent solutions in the reservoirs on the CD-type microfluidic device were sequentially introduced into the detection chamber by centrifugal force generated by the rotation of the microfluidic device. The variation of resonance wavelength was dependent on the refractive index of the sample solution. This CD-type SPR sensor was successfully used in an immunoassay of immunoglobulin A (IgA). The anti-IgA, blocking reagent, sample and washing solution in the reservoirs were sequentially introduced into the detection chamber by changing the frequency of rotation of the microfluidic device. IgA in the sample solution was adsorbed to the anti-IgA immobilized on the Au thin layer in the detection chamber and was then detected by the SPR sensor.