DOI： 10.1007/s44211-024-00588-6

PubMed

<p>Optical sensors (optodes) based on hydrophobic dyes and molecular recognition molecules have been applied to a variety of sensing applications, and are expected to be applied to medical diagnosis and life science research. In our laboratory, an enzyme-responsive dye liquid nanoemulsion (NE), which combines an NE-type optode and a novel dye liquid, was developed, and a significant reduction in response time compared to the conventional thin film type was achieved. However, because the above NE uses a fluorescent substrate that has an enzyme substrate site and a fluorescent site in one molecule, it has been difficult to examine the ratio of the substrate site to the fluorescent site, which is considered effective for further increasing sensitivity. Therefore, this study aimed to propose and demonstrate an enzyme-responsive NE-type optode in which the substrate site and the fluorescent site are introduced into NE as independent molecules. In this study, naphthol AS-TR phosphate was used as an alkaline phosphatase (ALP) substrate and acridine orange (AO) derivative, which is a cationic dye that emits strong fluorescence in a hydrophobic environment, was used as a fluorescent dye. When NE containing only ALP-responsive molecules was mixed with the ALP sample, a change in the absorption spectrum of naphthol AS-TR phosphate was observed, confirming the enzymatic reaction. Subsequently, when ALP-responsive NE containing the AO/TFPB ion pair was mixed with the ALP sample, a decrease in fluorescence intensity was observed. Naphthol AS-TR phosphate was dephosphorylated by the enzymatic reaction at the oil-water interface, and the cationic dye AO moved to NE/aq. interface to balance the electrical charge in NE. Furthermore, investigating the AO/TFPB ion pair ratio for ALP-responsive molecules, it was found that it was possible to increase the sensitivity by examining the conditions. Based on these results, the development of an enzyme-responsive NE-type optode with a new response mechanism was successfully demonstrated for the first time.</p>

DOI： 10.2116/bunsekikagaku.73.351

小分子と複合体を形成した核酸の構造変化を、ポリマー溶液を用いたキャピラリー分子篩電気泳動法(CSE)で評価した。モデル標的および核酸としては、L-チロシンアミド(Tyr-Am)およびTyr-Amに特異的に結合するDNAの一種であるアプタマーを選択した。CSEは、篩マトリックスとしてヒドロキシプロピルセルロースを含むバックグラウンド溶液(BGS)を満たしたキャピラリーを使用した。CSEのBGSにTyr-Amまたはチロシンを添加すると、Tyr-Am-アプタマー複合体の移動度差の比率は、Tyr-Amを添加していない遊離アプタマーと比較して増加した。他のアミノ酸またはその類似体を添加した場合には、電気泳動的移動度に明らかな変化は示されないか、または減少した。これらの結果から、本法は小分子を標的とする核酸の構造変化の評価に適用できると考えられた。

DOI： 10.1039/d3an02165c

PubMed

DOI： 10.1007/s44211-024-00524-8

PubMed

<p>A two-dimensional electrophoresis method, involving involves isoelectric focusing in the first dimension and polyacrylamide gel electrophoresis in the second dimension, is commonly used for the separation and analysis of protein mixtures. However, conventional methods have some drawbacks such as complex experimental procedures, long analysis time, and high consumption of reagents. In this study, based on the knowledge gained from our previous research on digital electrophoresis, a cartridge device with heterogeneous functionalized gels arranged and integrated in a two-dimensional pattern was used. The fundamental evaluation using model proteins confirmed the feasibility of sample introduction using the developed devices, digital sieving electrophoresis, and digital isoelectric focusing using pH-buffered gels. Furthermore, the verification of two-dimensional digital electrophoresis using a cartridge device with multiple pH-buffered gels and molecular sieving gels demonstrated continuous two-dimensional digital separation by employing the single device.</p>

DOI： 10.2116/bunsekikagaku.72.369

タンパク質混合試料の分離分析法として,一次元目に等電点電気泳動,二次元目にポリアクリルアミドゲル電気泳動を行う二次元電気泳動法が一般的に利用されている.しかしながら,従来法では煩雑な実験操作と長い分析時間を要するほか,多量の試薬を消費する点が課題であった.そこで本研究では,これまでに開発してきたデジタル電気泳動に関する知見を基に,異種機能性ゲルが二次元的に配置・集積された平板型カートリッジデバイスを開発した.モデルタンパク質を用いた基礎評価の結果,作製したデバイスと治具を用いた試料導入,デジタル分子ふるい電気泳動,及びpH緩衝ゲルを用いたデジタル等電点電気泳動がそれぞれ可能であることが確認された.さらに,複数のpH緩衝ゲル及び分子ふるいゲルが配置・集積されたカートリッジデバイスを用いた二次元デジタル電気泳動について検証した結果,同一デバイス内での連続的二次元デジタル分離が実証された.(著者抄録)

DOI： 10.1007/s44211-023-00337-1

PubMed

DOI： 10.1039/d3ra03245k

PubMed

<p>Aptamers, which show properties of specific binding to their target molecules, are expected to become an alternative to antibodies for use as molecular recognition probes instead of antibodies. However, long and laborious selection procedures have hindered their application. To overcome the drawbacks, rapid and simple selection methods using a microscale electrophoretic filtration device have been developed. In the first study, the aptamers for immunoglobulin E were successfully selected by three cycle selection based on the developed method. To realize more rapid and simple selection, aptamer selection by single cycle procedure was demonstrated. It was confirmed that CD63 as a model target was successfully trapped by the capillary device partially plugged with a polyacrylamide hydrogel. A DNA library containing various sequences were then electrokinetically introduced into the device after trapping CD63, a large volume of DNAs bound with the trapped CD63. After washing and elution schemes, DNAs located in the eluents were enhanced by PCR, and then their sequences were analyzed by next generation sequencer. As a result, one of the obtained aptamer candidates shows similar motifs to the previously reported aptamer. Isothermal titration calorimetry verified the binding ability and specificity of the obtained candidate to CD63. Consequently, it was clarified that the developed method and device is applicable to the rapid and simple selection of aptamers using the single cycle procedure.</p>

DOI： 10.2116/bunsekikagaku.72.111

標的分子に対する特異的結合能を示す配列を持つ核酸であるアプタマーは,抗体に代わる分子認識素子としての応用が期待される分子である.しかしながら,従来のアプタマー選抜法では煩雑な実験操作を10～15サイクル繰り返す必要があるため,アプタマー関連研究におけるボトルネックとなってしまっている.本研究では,これまでに開発したミクロスケール電気泳動フィルタリングに基づくアプタマー選抜法を細胞外小胞マーカー(CD63)のアプタマー選抜へと応用した.その結果,簡便な電気泳動に基づく標的分子導入,核酸導入,結合した核酸の洗浄・回収からなるわずか1サイクルの選抜操作によって,CD63アプタマー候補が獲得された.また,等温滴定カロリメトリーの結果から,選抜されたアプタマー候補の一つがCD63に対する結合能を持ち,非標的分子のウシ血清アルブミンには応答しないことが確認された.(著者抄録)

DOI： 10.1007/s11696-022-02485-6

DOI： 10.18494/SAM4329

DOI： 10.1117/12.2666797

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

This study reports a novel aptamer selection method based on microscale electrophoretic filtration. Aptamers are versatile materials that recognize specific targets and are attractive for their applications in biosensors, diagnosis, and therapy. However, their practical applications remain scarce due to issues with conventional selection methods, such as complicated operations, low-efficiency separation, and expensive apparatus. To overcome these drawbacks, a selection method based on microscale electrophoretic filtration using a capillary partially filled with hydrogel was developed. The electrophoretic filtration of model target proteins (immunoglobulin E (IgE)) using hydrogel, the electrokinetic injection of DNAs to interact with the trapped proteins, the elimination of DNAs with weak interactions, and the selective acquisition of aptamer candidates with strong interactions were successfully demonstrated, revealing the validity of the proposed concept. Two aptamer candidates for IgE were obtained after three selection cycles, and their affinity for the target was confirmed to be less than 1 nM based on their dissociation constant (KD) values. Therefore, the proposed method allows for the selection of aptamers with simple operations, highly effective separation based on electrophoresis and filtration, and a relatively cheap apparatus with disposable devices.

DOI： 10.3390/molecules27185818

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

A quartz crystal microbalance (QCM) is a sensor that uses the piezoelectric properties of quartz crystals sandwiched between conductive electrodes. Localized surface plasmon resonance (LSPR) is an analytical technique that uses the collective vibration of free electrons on metal surfaces. These measurements are known as analysis techniques that use metal surfaces and have been applied as biosensors because they allow for the label-free monitoring of biomolecular binding reactions. These measurements can be used in combination to analyze the reactions that occur on metal surfaces because different types of information can be obtained from them. However, as different devices are used for these measurements, the results often contain device-to-device errors and are not accurately evaluated. In this study, we directly fabricated gold nanostructures on the surface of a QCM to create a device that can simultaneously measure the mass and refractive index information of the analyte. In addition, the device could be easily fabricated because nanoimprint lithography was used to fabricate gold nanostructures. As a proof of concept, the nanoparticle adsorption on gold nanostructures was evaluated, and it was observed that mass and refractive index information were successfully obtained without device-to-device errors.

DOI： 10.3390/mi13091430

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Surface-enhanced Raman scattering (SERS) is a technique used to distinguish the constitution of disease-related biomarkers in liquid biopsies, such as exosomes and circulating tumor cells, without any recognition elements. Previous studies using metal nanoparticle aggregates and angular nanostructures have achieved the detection of various biomarkers owing to strong hot spots and electromagnetic (EM) fields by localized surface plasmon resonance (LSPR). Although these SERS platforms enable significant enhancement of Raman signals, they still have some problems with the fabrication reproducibility of platforms in obtaining reproducible SERS signals. Therefore, highly reproducible fabrication of SERS platforms is required. Here, we propose the application of a polymer-based gold (Au) nanocone array (Au NCA), which extensively generates an enhanced EM field near the Au NCA surface by LSPR. This approach was experimentally demonstrated using a 785 nm laser, typically used for SERS measurements, and showed excellent substrate-to-substrate reproducibility (relative standard deviation (RSD) &lt; 6%) using an extremely simple fabrication procedure and very low laser energy. These results proved that a Au NCA can be used as a highly reproducible SERS measurement to distinguish the constitution of biomarkers.

DOI： 10.3390/mi13081182

PubMed

Publishing type：Research paper (scientific journal)  

In this work, by combining a photonic crystal (PhC) and highly luminescent material, we developed an extremely sensitive PhC sensor that allows detection by single wavelength fluorescence intensity change in the visible wavelength region. We fabricated a titanium dioxide (TiO2) PhC containing quantum dots (QDs) inside the PhC structure and confirmed that the fluorescence intensity of the QDs was enhanced. Fluorescence enhancement occurs because of the total reflection and light confinement of the QD fluorescence inside the TiO2 PhC structure. The response to changes in the surrounding refractive index and protein adsorption was confirmed. Furthermore, for biosensing applications, troponin T (TnT), an acute myocardial marker, was determined using an antigen -antibody reaction. It was found that a TnT level of 100 fg/mL, which is considerably less than the cutoff value of 100 pg/mL, was successfully detected. The lower limit of detection (LOD) was found to be 58.2 fg/mL, which is 1/40 of that of the conventional enzyme-linked immunosorbent assay (ELISA) method (LOD: 2 pg/mL). It was demonstrated that the present TiO2 PhC-containing QDs can be used as a sensor device for the highly sensitive detection and determination of disease biomarkers.

DOI： 10.1016/j.mee.2022.111842

DOI： 10.1007/s44211-022-00101-x

PubMed

デジタル電気泳動法は、各種機能性ヒドロゲルに対する試料分子の通過・非通過を利用した分離・濃縮法として近年注目されているが、ヒドロゲル充填デバイス作製の煩雑さが大きな課題であった。そこで、本研究では、簡易デバイスを用いたデジタル電気泳動技術の開発と、機能性ヒドロゲルによるタンパク質の分離・濃縮評価のため、異種ポリアクリルアミドゲル連続充填キャピラリーデバイスを作製し、デジタル分子ふるい電気泳動の基礎評価を行った。その結果、プレポリマー溶液中アクリルアミド総濃度(%T)と通過・非通過タンパク質の分子量との相関が明らかとなった。また、濃縮効率評価の結果、最大で約180倍の高感度化が確認された。さらに、5、15、25%Tポリアクリルアミドゲル連続充填デバイスを用いたデジタル分子ふるい電気泳動によって、分子量が異なるタンパク質の分離と濃縮が同時に達成された。以上の結果から、作製したデバイスを用いた簡便・高分離能・高感度なデジタル電気泳動が実証された。(著者抄録)

Publishing type：Research paper (scientific journal)  

Metal nanostructures have attracted attention because of their unique optical characteristics derived from localized surface plasmon resonance. In this study, gold nanoholes with edge structures were designed and characterized to possess two plasmon bands at different wavelengths. These bands were assigned to the plasmon modes that generated a concentrated electric field at the tip of the individual edge structures. Furthermore, wavelengths of these bands were independently tuned from the visible to near-infrared wavelengths with shape and size of the gold nanoholes and edge structures as variables. To demonstrate wavelength tuning, a gold nanostructure with edge plasmon bands at wavelengths of approximately 785 and 1064 nm (typical laser wavelengths for surface-enhanced Raman scattering and optical tweezers, respectively) was successfully obtained via optical simulation. The results indicate that the structure designed in this study provides a guideline for the design of optical devices with efficient multiple optical functions.

DOI： 10.1016/j.rinp.2022.105541

Publishing type：Research paper (scientific journal)  

Highly sensitive optical sensors for label-free detection of DNA must have high usability and wide applicability for achieving advances in the medical and biological science fields. However, current sensors do not simultaneously satisfy these criteria. Here, we developed a simple, cost-effective, and sensitive plasmonic-photonic hybrid sensor using gold nanorods as nanoantennae and a photonic crystal slab (PCS) as a microcavity. In this report, we performed simple microscopic detection and identification of DNA molecules with Alzheimer's disease-associated single nucleotide polymorphisms, which is an important indicator for early diagnosis. The PCS was fabricated using nanoimprint lithography; the gold nanorods were controllably coupled with the PCS. The device sensitivity was experimentally assessed by controlling the plasmonic-photonic coupling resonance, which was enabled by tuning the PCS-coupled AuNR amounts. The sensitivity was also investigated by theoretical modeling of the hybrid sensor, based on the temporal coupled mode theory (TCMT). The TCMT-based theoretical model well explained the experimental results. In a demonstration of ultrasensitive detection of DNA, the identification of single nucleotide mismatched DNA molecules (similar to 1 pM) and detection of several DNA molecules (similar to 1000), with a limit of detection of 5.9 aM, were successfully achieved. This sensor may thus have wide applications for DNA analysis.

DOI： 10.1016/j.snb.2022.131747

DOI： 10.1039/d2an00115b

PubMed

DOI： 10.3390/bios12040200

PubMed

DOI： 10.3390/mi13020257

PubMed

DOI： 10.1541/ieejsmas.142.29

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Metal nanostructures exhibit specific optical characteristics owing to their localized surface plasmon resonance (LSPR) and have been studied for applications in various optical devices. The LSPR property strongly depends on the size and shape of metal nanostructures; thus, plasmonic devices must be designed and fabricated according to their uses. Nanoimprint lithography (NIL) is an effective process for repeatedly fabricating metal nanostructures with controlled sizes and shapes and require optical properties. NIL is a powerful method for mass-producible, low-cost, and large-area fabrication. However, the process lacks flexibility in adjusting the size and shape according to the desirable optical characteristics because the size and shape of metal nanostructures are determined by a single corresponding mold. Here, we conducted a re-shaping process through the air-plasma etching of a polymer's secondary mold (two-dimensional nanopillar array made of cyclo-olefin polymer (COP)) to modulate the sizes and shapes of nanopillars; then, we controlled the spectral characteristics of the imprinted plasmonic devices. The relationship between the structural change of the mold, which was based on etching time, and the optical characteristics of the corresponding plasmonic device was evaluated through experiments and simulations. According to evaluation results, the diameter of the nanopillar was controlled from 248 to 139 nm due to the etching time and formation of a pit structure. Consequently, the spectral properties changed, and responsivity to the surrounding dielectric environment was improved. Therefore, plasmonic devices based on the re-shaped COP mold exhibited a high responsivity to a refractive index of 906 nm/RIU at a wavelength of 625 nm.

DOI： 10.3390/mi12111323

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

An enzyme-responsive fluorescent nanoemulsion (NE) based on lipophilic dye liquid (LDL) was developed for alkaline phosphatase (ALP). The response mechanism of the NE involved enzymatic reactions and simultaneous extraction of anions. The LDL-based NE exhibited 3.8 times higher sensitivity than plasticizer-based conventional NE. Detection limit and response range were 2.7 (U L-1) and 5-50 (U L-1), respectively. The response time was reduced to less than half that of the LDL-based membrane.

DOI： 10.1039/d1an00447f

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

Aptamers, single-stranded DNAs/RNAs with a strong and specific interaction towards a target molecule, have wide applications in the fields of medicine and biosensors. In conventional aptamer selection methods, it is difficult to obtain "preorganized" and/or "induced-fit" type of aptamers selectively. In this study, separation and fractionation of single-stranded DNAs with/without stable preorganized structures were carried out using capillary sieving electrophoresis. The fractionated DNAs showed different mobilities and thermodynamic stabilities of their secondary structures; this outcome is deemed to be necessary for the synthesis of novel aptasensors with a desirable sensing mechanism.

DOI： 10.2116/analsci.21C003

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

DOI： 10.2116/analsci.21N011

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

DOI： 10.2116/analsci.21P067

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Analytical methods with fluorescence detection are in widespread use for detecting low abundance analytes. Here, we report a simple method for fluorescence signal amplification utilizing a structure of an azide-unit pendant water-soluble photopolymer (AWP) in a microchannel. The AWP is a poly(vinyl alcohol)-based photocross-linkable polymer, which is often used in biosensors. We determined that the wall-like structure of the AWP (AWP-wall) constructed in a microchannel functioned as an amplifier of a fluorescence signal. When a solution of fluorescent molecules was introduced into the microchannel having the AWP-wall, the fluorescent molecules accumulated inside the AWP-wall by diffusion. Consequently, the fluorescence intensity inside the AWP-wall increased locally. Among the fluorescent molecules considered in this paper, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) (DDAO) showed the highest efficiency of fluorescence signal amplification. We prepared a calibration curve for DDAO using the fluorescence intensity inside the AWP-wall, and the sensitivity was 5-fold that for the microchannel without the AWP-wall. This method realizes the improved sensitivity of fluorescence detection easily because the fluorescence signal was amplified only by injecting the solution into the microchannel having the AWP-wall. Furthermore, since this method is not limited to only the use of microchannel, we expect it to be applicable in various fields.

DOI： 10.1021/acsomega.1c00057

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Optical sensors based on solvent polymeric membranes have the potential to measure analytes present in an aqueous solution through the development of a tailored method for a specific target. However, limits in the concentrations of the component dyes have prevented improvements in sensitivity. We propose a Förster resonance energy transfer (FRET)-based fluorescence amplification system for ion-selective optical sensors using a highly fluorescent liquid material composed of a lipophilic phosphonium cation and a pyrene modifying sulfonate anion ([P66614][HP-SO3]), as both the plasticizer and donor, in addition to a combination of the lipophilic phosphonium cation and the fluorescein dodecyl ester anion ([P66614][12-FL]) as the fluorescent sensing dye acceptor. For ion extraction-based sensing, the donor and acceptor were retained in the plasticized PVC membrane with negligible leaching upon exposure to acidic and basic aqueous solutions. Systematic investigation of the donor and acceptor ratios clarified the effect of the amplification factor and the sensitivity of the sensor. At an acceptor doping level of 0.5 mol % (vs donor), an approximately 22-fold higher sensitivity was obtained compared to that of a conventional PVC membrane optical sensor. During ion measurement based on the coextraction of protons and anions, selectivity following the Hofmeister order was observed, which was controlled by the addition of ionophores. The proposed FRET system based on a lipophilic fluorescent liquid material has the potential to significantly improve the sensitivities of optical sensors using solvent polymeric membranes with high selectivities for various target analytes.

DOI： 10.1021/acs.analchem.0c05007

PubMed

本研究では、インクジェットプリンティングを用いて一つのポリジメチルシロキサン(Poly(dimethylsiloxane)、PDMS)製マイクロ流路内に2種類の反応試薬を固定化した1ステップ均一系競合型バイオアッセイマイクロデバイスを開発した。インクジェットプリンターは試薬をナノリットルサイズの液滴として正確な量を位置選択的に吐出するため、互いに反応する2種類の試薬を混ざることなく微少空間内に独立して固定化できる。そのため、先行研究でデバイスを作製する際に課題となった、相互に反応する試薬を固定化した二つのマイクロ流路を精密に組み合わせる操作が不要となる。本研究ではその一例として、ビオチン固定化酸化グラフェン(BG)と蛍光標識ストレプトアビジン(F-SA)を1本のマイクロ流路内に固定してビオチンアッセイマイクロデバイスを試作し、基礎検討として試薬の固定化位置や試薬及び添加剤種の評価を行った。その結果、液滴を流路壁面寄りに吐出すると再現性よく流路底面の両サイドに試薬固定できること、添加剤としてトレハロースが適すること及び、試料導入操作のみでの1ステップ検出で、検出下限は0.68ng mL-1となり、先行研究と比較して約1/600倍改善することを明らかにした。(著者抄録)

DOI： 10.2116/bunsekikagaku.70.125

Publishing type：Research paper (scientific journal)  

© 2020, The Author(s). An ionic liquid-based thin (~ 1 µm) colorimetric membrane (CM) is a key nano-tool for optical ion sensing, and a two-dimensional photonic crystal slab (PCS) is an important nano-platform for ultimate light control. For highly sensitive optical ion sensing, this report proposes a hybrid of these two optical nano-elements, namely, a CM/PCS hybrid. This structure was successfully fabricated by a simple and rapid process using nanoimprinting and spin-coating, which enabled control of the CM thickness. Optical characterization of the hybrid structure was conducted by optical measurement and simulation of the reflection spectrum, indicating that the light confined in the holes of the PCS was drastically absorbed by the CM when the spectrum overlapped with the absorption spectrum of the CM. This optical property obtained by the hybridization of CM and PCS enabled drastic improvement in the absorption sensitivity in Ca ion sensing, by ca. 78 times compared to that without PCS. Experimental and simulated investigation of the relation between the CM thickness and absorption sensitivity enhancement suggested that the controlled light in the PCS enhanced the absorption cross-section of the dye molecules within the CM based on the enhanced local density of states. This highly sensitive optical ion sensor is expected to be applied for micro-scale bio-analysis like cell-dynamics based on reflectometric Ca ion detection.

DOI： 10.1038/s41598-020-73858-8

PubMed

Publishing type：Research paper (scientific journal)  

Herein, a fully lipophilic ionic liquid (IL) comprising a lipophilic fluorescein anion and a trihexyltetradecylphosphonium cation was synthesized and used as the plasticizer for a plasticized poly(vinyl chloride) (PVC) membrane optode. Systematic investigation of the alkyl chain length of the fluorescein anion proved the significance of lipophilicity for obtaining the reversible absorbance measurements. A PVC membrane fabricated with the synthesized lipophilic IL was observed to comprise an unusually high dye concentration (915 mmol kg-1) and exhibited good sensitivity as well as response time in its sensor performance. The sensitivity of the presented PVC membrane was 26-fold higher than that of a conventional optode membrane with the same membrane thickness and the same lipophilic dye of typical dye content (1 wt%). The response time was observed to be >120-fold faster by using a significantly thinner PVC membrane (approx. 140 nm). Heparin is known to be a polyanionic anticoagulant, and the presented PVC membrane exhibited an extremely fast response (20-150 seconds) to the heparin in diluted serum within the required concentration region. Thus, the lipophilic IL-based dye could significantly improve the sensor performance in conventional optodes, especially for an analyte showing slow diffusion, such as macromolecular heparin.

DOI： 10.1039/d0an00335b

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1039/d0an90052d

PubMed

Publishing type：Research paper (scientific journal)  

© 2020 by the authors. Exosomes, a type of extracellular vesicle with a diameter of 30-150 nm, perform key biological functions such as intercellular communication. Recently, size sorting of exosomes has received increasing attention in order to clarify the correlation between their size and components. However, such sorting remains extremely difficult.Here, we propose to sort their size by controlling their electrokineticmigration in nanochannels in a micro-nanofluidic device, which is achieved by tuning the thickness of the electric double layers in the nanochannels. This approach was demonstrated experimentally for exosomes smaller than 250 nm. Using different running buffer concentrations (1 × 10-3, 1 × 10-4, and 1 × 10-5 M), most of the exosomes larger than 140, 110, and 80 nm were successfully cut off at the downstream of the nanochannels, respectively. Therefore, it is clarified that the proposed method is applicable for the size sorting of exosomes.

DOI： 10.3390/MI11050458

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

In this study, we report an inkjet printing-based method for the immobilization of different reactive analytical reagents on a single microchannel for a single-step and homogeneous solution-based competitive immunoassay. The immunoassay microdevice is composed of a poly(dimethylsiloxane) microchannel that is patterned using inkjet printing by two types of reactive reagents as dissolvable spots, namely, antibody-immobilized graphene oxide and a fluorescently labeled antigen. Since nanoliter-sized droplets of the reagents could be accurately and position-selectively spotted on the microchannel, different reactive reagents were simultaneously immobilized onto the same microchannel, which was difficult to achieve in previously reported capillary-based single-step bioassay devices. In the present study, the positions of the reagent spots and amount of reagent matrix were investigated to demonstrate the stable and reproducible immobilization and a uniform dissolution. Finally, a preliminary application to a single-step immunoassay of C-reactive protein was demonstrated as a proof of concept.

DOI： 10.3389/fchem.2020.612132

PubMed

Publishing type：Research paper (scientific journal)  

© The Japan Society for Analytical Chemistry. Herein, we describe the development of a novel material, "enzyme-responsive fluorescent ionic liquid", which enabled a highly sensitive detection of alkaline phosphatase (ALP). We prepared a plasticized poly(vinyl chloride) (PVC) membrane using this new material as a plasticizer and quantified ALP in aqueous solutions. Preliminary results suggested that the PVC membrane responded to ALP at an interface between the membrane and the sample solution with anion extraction to maintain electroneutrality in the membrane phase. The developed PVC membrane showed an approximately six-times higher sensitivity than the conventional membrane, thereby demonstrating highly sensitive ALP detection. These results suggested the potential applicability of the proposed membrane for highly sensitive protein detection by using ALPlabeled antibodies.

DOI： 10.2116/analsci.19C013

PubMed

Publishing type：Research paper (scientific journal)  

© The Royal Society of Chemistry 2019. A highly lipophilic ionic liquid (IL) consisting of stoichiometrically equal amounts of two purely functional chemical sensing molecules, an anionic calcium ionophore and a cationic dye, was synthesized for the first time, and used as a component for a poly(vinyl chloride)-based or neat liquid membrane optical sensor. This is the first report of an IL consisting of purely functional chemical sensing molecules, which is completely different from the previously reported ionic liquids typically consisting of imidazolium or other lipophilic cations. Since the present IL contained an extremely high concentration of dyes, the IL-based sensor showed dramatically enhanced sensitivity (13 times higher compared to that of a conventional sensor), and fully reversible and selective response to Ca2+. Preliminary investigation on the unique response characteristics of the present liquid membrane IL-based sensor was performed with the responses to Ca2+ and Na+.

DOI： 10.1039/c9an01769k

PubMed

Publishing type：Research paper (scientific journal)  

© 2019 Elsevier B.V. Metal nanostructures have great potential for optical label-free biosensors based on localized surface plasmon resonance (LSPR). The sensitivity of a metal nanostructure-based label-free biosensor (i.e., plasmonic sensor) depends on its plasmonic properties, which suffer a decrease in sensitivity by energy losses in the metal material. Here, we demonstrate an approach to improve the plasmonic properties of metal nanostructures by controlling the carrier density in the base polymer material using titanium nitride (TiN). It is expected that the light energy absorbed by TiN is converted into excitons, and it will assist LSPs in the metal nanostructure; thus, the losses of the metal material are compensated by the excitons excited in TiN. We designed a TiN-contained polymer-metal/core-shell structured nanocone array (NCA), comprising TiN nanoparticles (NPs) in a polymer core and metal shell (Au or Ag), and realized improvement of the refractive index (RI) sensitivity of a label-free biosensor by optimizing the TiN-contained polymer composition. As a result, the TiN-contained polymer-metal NCA, with a TiN NP concentration of 10 wt% in the polymer core, had a 1.5-fold higher RI sensitivity than that of the same NCA without TiN NPs. The results of the resistance measurement of the metal surface with the TiN NP-contained polymer (10 wt%) under light exposure suggest the conversion of exposed light into LSPs of metal via TiN. It is suggested that plasmonic properties and sensor performances can be improved by the presented approach. Moreover, in DNA hybridization detection, an extremely low limit of detection of 117.5 fM was achieved.

DOI： 10.1016/j.snb.2019.126932

Publishing type：Research paper (scientific journal)  

Copyright © 2019 American Chemical Society. The refractive index sensitivity of a localized surface plasmon resonance (LSPR) sensor is correlated to an enhanced local electromagnetic (EM) field originating from noble metal nanostructures. Here, we demonstrated that extensive EM field enhancement by a gold (Au) nanocone array (AuNCA) allowed highly sensitive and label-free detection of biomolecules in the visible wavelength spectrum. The AuNCA consisted of a polymer core and an Au shell, which was fabricated by using simple and inexpensive nanoimprint lithography. Under LSPR excitation, AuNCA absorbs visible light of a specific wavelength and extensively enhances the EM field near its surface. It was shown that AuNCA had high refractive index sensitivity (417.5 nm/RIU) because of the large distribution of the enhanced EM field, covering a large surface of the NCA. Moreover, in DNA hybridization detection, a very low limit of detection of 161 fM was achieved, and 1-base mismatch DNA was successfully discriminated by using AuNCA.

DOI： 10.1021/acsanm.9b00930

Publishing type：Research paper (scientific journal)  

© 2019 Optical Society of America. In past studies, liquid crystal (LC)-based immunoassays were accomplished by fabricating an LC cell with two pieces of glass slides after immunobinding, which makes the determination of the immunoassay not in real-time and requires trained personnel. Herein, we developed the LC-based immunoassay by using rectangular capillaries as the substrate for immunobinding. The inner surface of rectangular capillaries was decorated with a long alkyl saline, dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP), followed by immobilization of human serum albumin (HSA) as the probe. In this situation, the orientation of LC was homeotropic and dark LC image was observed under polarized light. When the solution containing anti-human serum albumin (anti-HSA) were dispensed into the capillary through capillary action, the specific immunobinding between HSA and anti-HSA formed an immunocomplex on the inner surface of capillary, which disrupted the original orientation of LC and led to a dark-to-bright transition of the LC images. The quantification of anti-HSA can be achieved by measuring the length of the bright LC image in the rectangular capillary. By using this immunoassay, the limit of detection (LOD) for anti-HSA is 1 μg/mL, and it did not respond to HSA and anti-human immunoglobulin G (anti-h-IgG). On the other hand, the diversity of the LC-based immunoassay can be extended for HSA detection when we immobilized anti-HSA in the capillary. Because the post-fabrication of LC cell was waived by using rectangular capillaries to develop the LC-based immunoassay, it is more convenient for users to handle and collect more reliable data. Moreover, the results of the immunoassay were visualized through naked-eye and could be recorded by a smartphone; it is more suitable for portable and point-of-care applications.

DOI： 10.1364/OE.27.017080

PubMed

Publishing type：Research paper (scientific journal)  

© 2018 Elsevier B.V. In this study, we developed a novel particle-type material comprised of graphene and polyethylene glycol (PEG) to solve the slow response problem of the previously reported microdevice for single-step immunoassay. Characterization of these graphene/PEG hybrids (GPH) was carried out using a transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, and fluorescence analysis. We found that PEG attached to graphene surfaces behave like molecular sieves to separate free fluorescently-labeled antibodies from antigen-bound antibody immunocomplexes. The free antibody is PEG-permeable and directly adsorbs onto the graphene surface, where fluorescence is quenched by fluorescence resonance energy transfer. On the contrary, immunocomplexes are PEG-impermeable; thus, as the concentration of the antigen increases, the concentration of PEG-impermeable immunocomplexes also increases; consequently, increasing the fluorescence intensity of the solution. After characterizing GPH, single-step immunoassay microdevices were prepared by separately immobilizing the GPH and fluorescently-labeled antibodies on two independent microchannel arrays and combining them. Finally, we tested the proposed immunoassay microdevices with various known concentrations of C-reactive protein (CRP), an inflammatory marker protein, and successfully measured CRP concentration within an approximate time of 2 min by monitoring the fluorescence intensity change.

DOI： 10.1016/j.flatc.2018.12.003

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

© 2019 The Japan Society for Analytical Chemistry. In this work, ionic liquid materials composed of pH indicator dyes were synthesized and used as a plasticizer for a plasticized PVC membrane-based ion-selective optode. We chose several triphenylmethane dyes and synthesized ionic liquids with those by an ion-exchange procedure, and found that an ionic liquid composed of bromothymol blue (BTB) realized the highest ion-exchange ratio of approximately 100% (highest concentration of dye). The absorbance response of the PVC membrane using a BTB-based ionic liquid was decreased by ca. 37% using the repetitive introduction of acid and base solutions, and reached to exhibit a stable and reproducible response. The concentration of dye was 20~70-times higher than that of the conventional ones. In addition, the PVC membrane was applied for anion sensing based on the coextraction of protons and anions in aqueous solutions, and it was found that the ion selectivity followed the Hofmeister series. In the future, the present ionic liquid material is expected to be applied for highly-selective and sensitive ion-selective optodes by the modification of functional groups such as ion-recognition or reaction with the target molecule.

DOI： 10.2116/bunsekikagaku.68.945

Publishing type：Research paper (scientific journal)  

© 2018 In this study, we developed a polymer/TiO2 hybrid two-dimensional photonic crystal (2D-PhC) optical ion sensor that shows fluorescence enhancement based on the matching of the fluorescence wavelength and the confinable wavelength of a 2D-PhC. The polymer/TiO2 hybrid PhC was fabricated from a TiO2 pillar array, where the interspace was filled with a polymer containing a fluorescent dye. We developed a method of fabricating the TiO2 pillar array based on nanoimprint lithography using liquid phase deposition. The polymer/TiO2 hybrid PhC was fabricated on a poly(ethylene terephthalate) (PET) film. Thus, it can be easily cut because of its high flexibility and used for ion sensing applications. By using a fluorescent dye-containing polymer (poly(methyl methacrylate), PMMA) for the fabrication of the polymer/TiO2 hybrid PhC, the fluorescence intensity was drastically (25 times) increased. Subsequently, we used an ion-sensing polymer composed of plasticized poly(vinyl chloride) (PVC), a fluorescent dye, and ion recognition elements in the polymer/TiO2 hybrid PhC to enable its use as a highly sensitive ion sensor. As a result, the ion sensor based on the plasticized-PVC/TiO2 hybrid PhC exhibited four times greater fluorescence intensity compared with that based on the plasticized PVC without the PhC structure.

DOI： 10.1016/j.snb.2018.05.004

Publishing type：Research paper (scientific journal)  

© 2018 by the authors. In this paper, we have proposed a polymer-based photonic crystal (PhC) resonator, with multiple sizes of cavities, and a waveguide to be used as highly sensitive optical sensor components. Properties of the proposed PhC were simulated by the finite-difference time-domain method, and the polymer-based PhC resonator and waveguide were fabricated on a photoresist (polymer) by electron beam lithography, which was prepared on an Au-layer-deposited Si substrate. We detected the resonant light that penetrated through the waveguide and was trapped in the PhC resonator. Optical characteristics of the fabricated PhC were evaluated by detecting the polymer layer deposition process by using the layer-by-layer (LbL) method to deposit polymer layers. As a result, by using an optimized design of a polymer-based PhC resonator with a long cavity (equivalent to a defect of three holes), the PhC structure changes caused by LbL deposition lead to changes in resonant light wavelength (peak shift: 5.26 nm/layer). Therefore, we suggest that a PhC resonator and a waveguide is applicable as an optical sensor.

DOI： 10.3390/mi9080410

Publishing type：Research paper (scientific journal)  

© The Japan Society for Analytical Chemistry. A novel method for the intact immobilization of a very-thin and soft PVC membrane on a convex-shaped poly(dimethylsiloxane) (PDMS) surface is described. The present method using PVA film as a sacrificial layer allowed successful immobilization of an intact PVC membrane using an ionic liquid-based dye on only the convex-shaped PDMS surface without any deformation or increase of the inhomogeneity. In addition, two different kinds of PVC membranes were successfully immobilized simultaneously toward multiplexed detection.

DOI： 10.2116/analsci.18C008

PubMed

Publishing type：Research paper (scientific journal)  

© 2017 Wiley Periodicals, Inc. We fabricated 1 STEP biomarker protein analysis device using silver nanoparticles (AgNPs)-contained hydrogel and reagent-immobilized cartridge. Specific color change of AgNPs based on localized surface plasmon resonance (LSPR) was applied to develop the novel colorimetric protein analysis device. In this report, we constructed this device by four components, AgNPs-contained hydrogel, glucose oxidase (GOx) antibody release pad, glucose release pad, and antibody-immobilized membrane. AgNPs-contained hydrogel detected 0.001 to 10 μg/mL GOx-antibody. AgNPs-contained hydrogel absorbance became altered 90.0% GOx release pad as compared to GOx-antibody solution under the reaction time 60 min. AgNPs-contained hydrogel absorbance became altered 98.4% glucose release pad as compared to glucose solution under the reaction time 60 min. We applied AgNPs-contained hydrogel and antibody-immobilized membrane to detect human immunoglobulin G (IgG). As a result, we found these could detect 1 μg/mL or lower concentration of human IgG.

DOI： 10.1002/ecj.11960

Publishing type：Research paper (scientific journal)  

In this study, polyvinylpyrrolidone) (PVP)-coated silver nanoparticles were used as a chromogenic substrate for the establishment of a colorimetric enzyme-linked immunosorbent assay (ELISA) based 011 localized surface plasmon resonance (LSPR) called "plasmonic ELISA". The PVP-coated silver nanoparticles exhibit a specific color due to the LSPR. In addition, the LSPR absorbance strength of the PVP-coated silver nanoparticles is changed by the aggregation of the silver nanoparticles which depends 011 the hydrogen peroxide concentration. From these changes in the LSPR absorbance strength in relation to hydrogen peroxide concentration, plasmonic ELISA was established using a glucose oxidase (GOx)-conjugated secondary antibody and glucose for producing hydrogen peroxide by an enzymatic reaction. Furthermore. 011 the basis of plasmonic ELISA. a cartridge-based single-step plasmonic ELISA device was fabricated for the realization of a wash-free ELISA system. This cartridge-based ELISA system is constructed with a GOx-Antibody (GOx-conjugated secondary antibody) release pad. a primary antibody immobilized membrane, a glucose release pad, and a PVP-coated silver nanoparticle-containing hydrogel. By using this construction, the antigen concentration can be determined by introducing only a sample solution. As a result, with this cartridge-based ELISA system, the antigen-Antibody reaction was successfully detected at different concentrations of antigen (0.1-10 μg/ml).

DOI： 10.18494/SAM.2017.1642

We fabricated a titanium dioxide (TiO<inf>2</inf>)-based photonic crystal (PhC) using liquid phase deposition (LPD) to develop highly sensitive optical biosensors. The optical characteristics of the PhCs in the visible region were sensitive to the change in the refractive index of the surrounding medium due to an antigen–antibody reaction; thus, applications using the optical biosensor are expected to be highly sensitive. However, a base material with a high refractive index is indispensable for the fabrication of the PhC. Here, TiO<inf>2</inf>, which has optical transparency in the visible region, was selected as the high refractive index base material. The present LPD method allowed fabrication using low-cost apparatus. Furthermore, the mild conditions of the LPD method led to formation of TiO<inf>2</inf>-based PhC with fewer crack structures. Finally, the anti-neuron-specific enolase antibody was immobilized onto the TiO<inf>2</inf>-based PhC surface, and 1–1000 ng/mL of the neuron-specific enolase antigen was successfully detected.

DOI： 10.7567/JJAP.55.08RE01

CiNii Article

Publishing type：Research paper (scientific journal)  

© The Royal Society of Chemistry. A single-step, easy-to-use enzyme immunoassay capillary sensor, composed of functional multilayer coatings, was developed in this study. The coatings were composed of substrate-immobilized hydrophobic coating, hydrogel coating, and soluble coating containing an enzyme-labeled antibody. The response mechanism involved a spontaneous immunoreaction triggered by capillary action-mediated introduction of a sample antigen solution and subsequent separation of unreacted enzyme-labeled antibodies and antigen-enzyme-labeled antibody complexes by the molecular sieving effect of the hydrogel. An enzyme reaction at the substrate-immobilized hydrophobic coating/hydrogel coating interface resulted in a protein-selective fluorescence response. An antigen concentration-dependent response was obtained for diagnostic marker protein samples (hemoglobin A1c (HbA1c), 7.14-16.7 mg mL-1; alpha-fetoprotein (AFP), 1.4-140 ng mL-1; C-reactive protein (CRP), 0.5-10 μg mL-1) that cover a clinically important concentration range. The successful measurement of CRP in diluted serum samples demonstrated the application of this capillary sensor. This journal is

DOI： 10.1039/c4an01781a

PubMed

Publishing type：Research paper (scientific journal)  

The caspase-3 inhibitor assay, which is important for drug development was successfully integrated into a "single step" by solving the immobilization problem of enzymes with low activity. Here we used the soluble polyethylene glycol (PEG) coatings for enzyme immobilization to the concave-shaped PDMS surface, and substrate immobilization to the convex-shaped PDMS surface. Then these PDMS structures were combined to form a capillary-structure, which we call a combinable poly (dimethylsiloxane) (PDMS) capillary (CPC) sensor, enabling simultaneous immobilization of two different reactive reagents such as enzymes and fluorescent substrates. The present sensor was disposable, and the fluorescence response was simply obtained by introducing the sample solution by capillary action. The caspase-3 activity was able to be maintained at approximately 90% under -80 °C storage conditions even after 5 months. Importantly, the total reaction time was reduced from an hour to 3-5 min, and simultaneous acquisition of multiple data sets required to determine IC50 value was also successfully achieved using the CPC sensor array. © 2014 The Royal Society of Chemistry.

DOI： 10.1039/c3ra46976j

Publishing type：Research paper (scientific journal)  

A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme- conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL-1 linear range, is reported. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3lc50775k

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

To enhance sensitivity and facilitate easy sample introduction into a combinable poly(dimethylsiloxane) (PDMS) capillary (CPC) sensor array, PDMS was modified in bulk and on its surface to prepare "black" PDMS coated with a silver layer and self-assembled monolayer (SAM). India ink, a traditional Japanese black ink, was added to the PDMS pre-polymer for bulk modification. The surface was modified by a silver mirror reaction followed by SAM formation using cysteine. These modifications enhanced the fluorescence signals by reflecting them from the surface and reducing background interference. A decrease in the water contact angle led to enhanced sensitivity and easy sample introduction. Furthermore, a CPC sensor array for multiplex detection of serum sample components was prepared that could quantify the analytes glucose, potassium, and alkaline phosphatase (ALP). When serum samples were introduced by capillary action, the CPC sensor array showed fluorescence responses for each analyte and successfully identified the components with elevated concentrations in the serum samples. © The Royal Society of Chemistry.

DOI： 10.1039/c2lc21242k

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

A facile preparation method of a disposable capillary biosensor immobilizing a plasticized poly(vinyl chloride) (PVC) membrane ion-selective optode containing a lipophilic pH indicator dye and an ionophore, and a dissolvable poly(ethylene glycol) (PEG) membrane containing an enzyme is developed. The method involves the sequential introduction of membrane cocktail solution followed by air introduction. Repeating this process allowed facile preparation of a capillary biosensor comprised of two layer structure, which can be used for single-step biosensing just by sipping the sample with capillary action. Here a urea biosensor was prepared as a proof-of-concept. © 2010 The Chemical Society of Japan.

DOI： 10.1246/cl.2010.436

Publishing type：Research paper (scientific journal)  

A simple capillary enzymatic biosensor was developed. This was prepared by simply coating a dissolvable membrane containing enzyme/s on the inner wall of a square glass capillary. An easy measurement was carried out by capillary force sample introduction with concurrent enzyme release and a reaction with a certain substrate. Enzyme-release capillary (ERC) biosensors showed long-term storage stability of at least two weeks for a β-galactoside derivative and glucose. Moreover, this could be integrated on a capillary-assembled microchip (CAs-CHIP) to broaden its multiple analyte sensing potential for clinical diagnostic applications. 2009 © The Japan Society for Analytical Chemistry.

DOI： 10.2116/analsci.25.1025

PubMed

Publishing type：Research paper (scientific journal)  

Single-drop analysis of two different real sample solutions (2 μL) while simultaneously monitoring the activity of two sets of ten different proteases on a single microfluidic device is presented. The device, called a capillary-assembled microchip (CAs-CHIP), is fabricated by embedding square glass sensing capillaries (reagent-release capillaries, RRC) in the polydimethylsiloxane (PDMS) lattice microchannel, and used for that purpose. First, the performance reliability was evaluated by measuring the fluorescence response of twenty caspase-3-sensing capillaries on a single CAs-CHIP, and a relative standard deviation of 1.5-8.2 (% RSD, n∈=∈5 or 10) was obtained. This suggests that precise multiplexed protease-activity sensing is possible by using a single CAs-CHIP with multiple RRCs embedded. Then, using a single CAs-CHIP, real sample analysis of the activity of ten different caspases/proteases in cervical cancer (HeLa) cell lysate treated and untreated with the cell-death-inducer drug, doxorubicin, was simultaneously carried out, and a significant difference in enzyme activity between these two samples was observed. These results suggested the usefulness of the CAs-CHIP in the field of drug discovery. © 2008 Springer-Verlag.

DOI： 10.1007/s00216-008-2105-x

PubMed

Publishing type：Research paper (scientific journal)  

The experimental conditions of the sample delivery inside the reagent-release capillary-based capillary-assembled microchip (RRC-based CAs-CHIP) were optimized and the reagent release procedure in the RRC is discussed. Recently, our group introduced the basic concept of the "drop-and-sip" fluid handling technique (Anal. Chem., 2007, 79, 908). A microliter volume of sample solution is dropped on the inlet hole and is sipped into another hole, producing a sample plug flow in the main poly(dimethyl siloxane) (PDMS) channel, concurrently filling each sensing capillary that faces the main PDMS channel. However, the detailed evaluation of the successful sample delivery condition and the reagent release behavior in the RRC has not been fully discussed. Under our experimental conditions, ca. 0.6 - 2.4 s of sample plug-RRC contact time allowed the successful sample introduction into the RRC by capillary force without any reagent leakage or disturbance of the sample plug flow. On the other hand, reagent release behavior inside the RRC is governed by both convective and diffusive mass transport, which leads to a faster mixing time of the sample with reagents immobilized inside the RRC compared to that expected from the simple diffusion alone. 2008 © The Japan Society for Analytical Chemistry.

DOI： 10.2116/analsci.24.127

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

A general and simple implementation of simultaneous multiparametric sensing in a single microchip is presented by using a capillary-assembled microchip (CAs-CHIP) integrated with the plural different reagent-release capillaries (RRCs), acting as various biochemical sensors. A novel "drop-and-sip" technique of fluid handling is performed with a microliter droplet of a model sample solution containing proteases (trypsin, chymotrypsin, thrombin, elastase) and divalent cations (Ca2+, Zn2+, Mg2+) that passes through the microchannel with the aid of a micropipette as a vacuum pump, concurrently filling each RRC via capillary force. To avert the evaporation of the nanoliter sample volume in each capillary, PDMS oil is dropped on the outlet hole of the CAs-CHIP exploiting the capillary force that results in spontaneous sealing of all the RRCs. In addition, this high-speed sample introduction alleviates the possibility of protein adsorption and capillary cross-contamination, allowing a reliable and multianalyte determination of a sample containing many different proteases and divalent cations by using the fluorescence image analysis. Presented results suggested the possible application of this microchip in the field of drug discovery and systems biology. © 2007 American Chemical Society.

DOI： 10.1021/ac061245i

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

A capillary immunosensor is developed as a sensing unit of a capillary-assembled microchip (CAs-CHIP) that is geared towards simultaneous multiple immunoassay. It is prepared by subsequently treating the inner surface of a square glass capillary (internal dimension 50 μm) with 3-aminopropyltriethoxysilane -glutaraldehyde-protein A (APTES-GA-Protein A), which significantly reduced the non-specific adsorption. Consequently, accurate quantitative immunoassay measurement is demonstrated using human and chicken IgG as model samples, yielding a detection limit of about 1 ng mL-1 for both antigens with a total analysis time of about 60 min. The immunosensor also displayed interesting practical properties like long-term stability of at least a month at 10°C and minimal consumption of secondary labeled-antibody with one-million-fold dilution. It is expected that the integration of this fluorescent immunosensing unit into the CAsCHIP will enhance the analytical performance of the microchip through simultaneous multiple immunoassay in a single microfluidic device. This type of biochip may have a significant impact in clinical diagnostics and drug-screening applications.

Publishing type：Research paper (scientific journal)  

A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed. © Springer-Verlag 2006.

DOI： 10.1007/s00216-006-0331-7

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into β-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction. © 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chroma.2005.11.125

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Chemical syntheses using microchemical chips have been intensively investigated in recent years, but they have still not fully reached the stage of practical use. Combinatorial synthesis has been expected to be one of their useful applications. Benefits of using microchips, such as reduction in quantity of reagents and wastes, are thought to be suited for combinatorial syntheses, where numerous variations of products have to be synthesized each in small amounts. There are two modes of combinatorial syntheses using microchemical chips. One is sequential synthesis, in which reactant solutions are introduced sequentially in various combinations to a reaction microchannel. The other is parallel synthesis, where the number of reaction channels is the same as the number of final products, and starting materials are distributed to all the reaction channels in different combinations. Sequential synthesis has been considered as the more favorable way, because, especially when libraries of starting materials are large, numerous microchannels and complicated pipings for distribution of reagents to the channels are required for the parallel mode. Therefore, research on parallel microcombinatorial syntheses has been done only rarely, whereas there have been many reports of sequential microcombinatorial syntheses. Against this trend, our research group has taken an alternative path and tried to integrate both parallel reaction microchannels onto a glass microchip and channels for distributing reactant solutions to each reaction channel in a combinatorial mode. Microchips fabricated by laminating multiple layers of glass substrates have been used to construct three-dimensional microchannel networks necessary for mixing reagent flows in different combinations. We utilized low Reynolds number multi-phase laminar flow and developed micro-unit operations (MUOs). By combining MUOs in the multi-phase continuous laminar flows, microchemical systems were constructed. We called this micro-integration methodology "continuous-flow chemical processing (CFCP)", and by using it, a sequence of various operations in chemical syntheses could be integrated on a microchemical chip. Not just simple mixing of two reagents, which was most commonly done in microreactors, but also multi-step processes including other chemical operations, such as extraction and separation, could be carried out via CFCP. In the first part of this short review article, we explain CFCP with an example. Then, a microchemical chip with a three-dimensional microchannel network for a parallel reaction system is introduced. Finally, we present a case in which CFCP was realized in a three-dimensional microchannel network. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/qsar.200540002

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

A recent microfabrication technology opened the new application field such as micro total analysis systems (Micro TAS) or microreactors. These systems employed physical characteristics provided by the liquid microspace, involving short diffusion distance, large surface to volume ratio, or small heat capacity. In contrast to these works, we focused on the chemical functions of highly-selective molecular recognition molecules or functional dyes of which we have been developed, to be integrated into microfluidic chemical sensing systems. The present paper describes our recent results concerning chemical sensing systems based on microfluidic devices. Here, a multi ion sensing system based on the intermittent reagent pumping and ion-pair extraction, a new biosensing scheme based on the enzyme-immobilized polymer membrane, and a novel multi ion sensing scheme based on capillary-assembled microchip, are presented. © 2005 The Japan Society for Analytical Chemistry.

DOI： 10.2116/bunsekikagaku.54.267

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Part of collection (book)  

This chapter provides information on micro and nano-fabrication technologies that have been introduced into chemical and biotechnologies. It also explains molecular and energy transport between multiphase laminar flow in a micro channel. As for chemical synthesis, micro reactors with sub-millimeter-sized micro channels are now eagerly investigated. Though there are some examples of electrokinetically-driven flow control in micro reactors, pressure-driven flow control is more commonly used for micro reactors. The aim is to develop methodology to integrate as many chemical processes as possible on microchips and to make full use of characteristics of micro space. Therefore, pressure-driven flow and developed techniques to operate chemical processing in pressure-driven laminar flow in micro channels are selected and for this microchips made of glass are used. Glass vessels are commonly used in many chemical experiments because of their chemical inertness and optical transparency. In order to realize these micro chemical systems based on multiphase laminar flow, some stabilization techniques for the multiphase flow has been inevitable. A micro channel with guide structures and surface modification to stabilize multiphase flows has been used. The chapter also presents a micro integration methodology, continuous flow chemical processing (CFCP), based on spontaneous motion of molecules in multi-phase laminar flow network in micro channel circuits. © 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/B978-044451100-3/50015-2

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

In this paper, new "multiinformation dyes" (MIDs) with extended spectral sensitivity reporting on both changes in solvent polarity (solvatochromism) and in pH (halochromism) by changes in their visible absorption spectra, are presented. The merocyanine dyes 2,6-dibromo-4-[4-(1-dodecyl-4(1H)-pyridinylidene)-2-butenylidene]-2,5- cyclohexadien-1-one (KD-M301) and 2,6-dibromo-4-[4-(1-dodecyl-4(1H)-quinolinylidene)-2-butenylidene]-2,5- cyclohexadien-1-one (KD-M402) were designed, synthesized, and characterized regarding analytical applications. The spectral sensitivity for the MIDs was extended into the near-infrared (NIR) wavelength range (λmax = 800 nm in CHCl3 for KD-M402) and at the same time, the degree of solvent polarity-induced spectral shifts was strongly enhanced (Δλmax = 202 nm for KD-M402 between tetrahydrofuran (THF) and methanol), reaching similar values to the standard solvatochromic dye Dimroth-Reichardt's betaine on which the empirical ET(30) classification of solvent polarity is based. Compared to Dimroth-Reichardt's betaine dye, the molar absorption coefficient ε, is increased more than 10-fold (ε of KD-M402 in THF: 7.7 × 104 M-1 cm-1), allowing sensitive measurements at low concentrations of the dye. The dodecyl-substituted KD-M402 is suitable as a lipophilic pH indicator in ion-exchange type optical sensors (optodes) with optical detection in the near-infrared wavelength range. © 2003 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0003-2670(03)00200-9

Publishing type：Research paper (scientific journal)  

Here we report a design and synthesis of a chemically functional polymer membrane by an interfacial polycondensation reaction and multilayer flow inside a microchannel. Single and parallel dual-membrane structures are successfully prepared by using organic/aqueous two-layer flow and organic/aqueous/organic three-layer flow inside the microchannel followed by an interfacial polycondensation reaction. By using the inner-channel membrane, permeation of ammonia species through the inner-channel membrane is successfully achieved. Furthermore, horseradish peroxidase is immobilized on one side of the membrane surface to integrate the chemical transform function onto the inner-channel membrane. Here substrate permeation through the membrane and subsequent chemical transformation at the membrane surface are realized. The polymer membrane prepared inside the microchannel has an important role in ensuring stable contact of different phases such as gas/liquid or liquid/ liquid and the permeation of chemical species through the membrane. Furthermore, membrane surface modification chemistry allows chemical transformation of permeated chemical species. These methods are expected to lead to development of complicated and sophisticated chemical systems involving membrane permeation and chemical reactions.

DOI： 10.1021/ac025794+

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

A new fluid flow inside the microchannel was successfully developed. The flow created here involves segmented flow injection of plural organic phases into a microchannel followed by contact with a single aqueous phase to form stable organic-aqueous two-layer flow inside the microchannel. Fundamental study on the developed flow inside the microchannel was performed by monitoring the dye-doped segmented organic phases by thermal lens microscopy (TLM). Excellent repeatability and very small injection volume in developing segmented flow were realized. The new fluid flow created here is expected to allow us multi-ion sensing, which is not easily demonstrated by conventional ion sensor technology using a solvent polymeric membrane, by combining with neutral ionophore-based ion pair extraction using plural numbers of organic phases containing different ionophore molecules. Keywords : Segmented flow injection, Microchip, μ-TAS, Multi-ion, Multiphase flow. © 2003, The Institute of Electrical Engineers of Japan. All rights reserved.

DOI： 10.1541/ieejsmas.123.124

Publishing type：Research paper (scientific journal)  

An integrated multireactor system for 2 × 2 parallel organic synthesis has been developed on a single glass microchip. Three-dimensional channel circuits in the chip were fabricated by laminating three glass plate layers. The fabrication method is a straightforward extension of the conventional one, and topological equivalence for any three-dimensional circuits can be constructed easily with it. 2 × 2 phase-transfer amide formation reactions, which constitute a simple model for combinatorial synthesis, were successfully carried out on the microchip, and the integrity of the three-dimensional circuits was confirmed. Combinatorial chemistry with multi-microreactors, in conjunction with a high-throughput screening method based on μ-TAS technologies, is expected to provide an efficient tool for drug discovery.

DOI： 10.1039/b208382p

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

A series of ionophores for ammonium ion-selective electrodes was designed and synthesized, and their characteristics were examined. The design of the ionophores is based on two different strategies: (1) introduction of bulky blocking subunits (decalino groups) in 20- or 21-membered crown ethers (TD20C6 and TD21C6), the ring size of which is expected to be suitable for selective NH4+ recognition, as compared to the slightly smaller K+; and (2) preorganized tripodal ionophores based on a 6-fold substituted benzene ring in order to complementarily recognize the tetrahedral NH4+, in contrast to the spherical K+. Compared to nonactin, a natural product that is used as a representative NH4+ ionophore, the newly developed TD20C6 showed higher NH4+ selectivity over K+ while retaining the selectivity over Na+(log KNH4+,K+pot≡ -1.5 and log KNH4+,Na+pot = -2.5). On the other hand, a tripodal ionophore with pyrazole nitrogen atoms as NH4+ binding sites showed high NH4+/K+. selectivity but suffered from increased Ca2+ interference (log KNH4+,K+pot = -2.1 and log KNH4+,Ca2+pot = -1.6). As an overall conclusion, the cyclic ionophores TD19C6 and TD20C6 are the best ammonium-selective ionophores developed to date.

DOI： 10.1021/ac025713+

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Novel Mg2+ fluorescent molecular probes (KMG-20-AM and KMG-27-AM; where AM is an acetoxymethyl group) based on a coumarin possessing a charged β-diketone structure were designed and synthesized. These fluorescent probes produced a red shift from 425 to 445 nm in the absorption spectra after formation of a complex with Mg2+. The fluorescence spectra of these probes also showed a red shift from 485 to 495 nm and an increasing fluorescence intensity after formation of a complex with Mg2+. The optimum experimental conditions were excitation wavelength of 445 nm and a monitored wavelength of 500 nm, where these probes functioned as an indicator showing an image of increasing fluorescence in the presence of Mg2+. These probes showed a "seesaw-type" fluorescent spectral change with the isosbestic point at 480 nm due to the light excitation at 445 nm, which indicates that ratiometry can be used for the measurement. The molecular probes formed a 1:1 complex with Mg2+ and the dissociation constant (Kd) was 10.0 mM for KMG-20. The association constants of the probes with Mg2+ were ∼3 times higher than that with Ca2+, which showed that the selectivity of Mg2+ versus Ca2+ for these probes was over 200 times higher than that for commercially available Mg2+ fluorescent molecular probes such as mag-fura-2, Magnesium Green. As an application of these probes, intracellular fluorescent imaging of Mg2+ was demonstrated using a fluorescent microscope. After the addition of KMG-20-AM and KMG-27-AM into PC12 cells, a strong fluorescence was observed in the cytoplasm and a weak fluorescence in the nuclei region. After treatment with a high-K+ medium, the fluorescence intensity increased due to increasing intracellular Mg2+. The real image of Mg2+ release from the magnesium store was successfully observed with these Mg2+ fluorescent probes.

DOI： 10.1021/ac010914j

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Thermal lens microscope (TLM) can be a powerful tool for a detection method for integrated chemistry laboratory (ICL). In spite of great potential of ICL, its application has been restricted due to the lack of highly applicable and sensitive detection methods. TLM developed by our group meets requirements of detection method for ICL, such as high sensitivity, high spatial resolution, and wide applicability. In this review, the principle and characteristics of TLM are introduced and usefulness of TLM in ICL is demonstrated in some analytical applications.

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

The preparation and response features of a micrometer-sized lithium ion-selective optode based on a liquid membrane were examined. The optode membrane was a plasticized poly(vinyl chloride) (PVC)-based copolymer incorporating a lipophilic 14-crown-4 derivative as the neutral lithium ionophore and a dibromofluorescein derivative as the fluorescent anionic dye. The detection mode was a fluorescence change based on the ion-pair extraction/ion exchange principle caused by the fluorescent anionic dye and the lithium ionophore. The 5-μm-sized microoptode was prepared by the micropipette fabrication method and characterized by measuring the optical responses to Li+ concentrations with the time-resolved photon counting method. The microoptode responded to lithium ion concentrations of ~0.5 to ~500mM. The micrometer-sized lithium ion-selective microoptode was successfully obtained when a tailed ionophore was used. The anchor effect of the tailed ionophore was useful for the lithium ion-selective microoptode to resolve leaching of the ionophore, which is a significant problem in a microoptode based on a liquid membrane. This demonstration indicates that other ion-selective microoptodes can be obtained simply by replacing the tailed ionophore. (C) 2001 Elsevier Science B.V.

DOI： 10.1016/S0003-2670(00)01148-X

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Molecular transport across a water-organic liquid interface formed parallel to the side wall of a microchannel on microchips has been studied using methyl red as model compound and cyclohexane and n-octanol as organic solvents. The molecular transport was monitored by thermal lens microscopy and the kinetics of the process was studied for different concentrations and flow velocities. The influence of physico-chemical interactions between the model compound and organic solvents on liquid-liquid interface was demonstrated in the microspace and corresponding partition coefficients were calculated which confirmed the experimental results.

DOI： 10.1081/AL-100104916

Publishing type：Research paper (scientific journal)  

Novel Hg2+ receptors having two N-benzyloxyamide groups are synthesized, and their complexation ability toward metal cations is examined. The structure of the formed complexes was elucidated by ESI-MS and 1H NMR spectroscopy. Deprotonation of the amide groups allows the receptors to form stable complexes with Hg2+. As an application of this neutral Hg2+ complex, an anion-selective electrode combined with a cationic additive is prepared, and exhibits an anti-Hofmeister selectivity pattern with enhanced selectivity for the iodide anion. The overall selectivity pattern of the electrode is I- > ClO4- > SCN- ≈ Br- > salicylate- > NO2- > Cl- > NO3- ≫ HCO3- > SO42- > F- > H2PO4-.

DOI： 10.1039/b008978h

Publishing type：Research paper (scientific journal)  

The large specific interfacial areas and short molecular diffusion distances provided by glass microchips play important roles not only for effective phase-transfer synthetic reaction, but also for avoiding an undesirable side reaction. © 2001 The Royal Society of Chemistry.

DOI： 10.1039/b106494k

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

The preparation and response features of a micrometer-sized sodium ion-selective fiber optode based on a liquid membrane were described. The sensing membrane is a plasticized poly (vinyl chloride)-based copolymer with a neutral ionophore and an anionic dye. In order to fabricate a micrometer-sized fiber optode, a "micropipette fabrication method" was newly proposed to fix a liquid membrane-based optode on the small tip of an optical fiber probe. At the first stage of the investigation, it was found that the ionophore including a sodium ion in its cavity leached from the membrane phase. However, we have discovered that the problem can be resolved by using a "tailed" ionophore, which is an ionophore possessing a lipophilic long alkyl chain. The "'tail" of the ionophore functions as an "anchor", which prevents leaching of die ionophore from the membrane phase into the water phase. The anchor effect of the tailed ionophore was clearly demonstrated with 6 μm-sized sodium ion-selective optodes. In addition, the problem of fluorescence distortion due to photobleaching and solvent effect was resolved by a ratiometric calibration in which the sensor response is monitored by the spectral shift of the dual-emission fluorescence. The sensor response of an 8 μm-sized fiber optode having ratiometric calibration was examined and successfully explained by response theory. Our method gives a general preparation for ultrasmall ion-selective fiber optodes because other ion-selective optodes can be obtained simply by replacing the tailed ionophore.

DOI： 10.2116/bunsekikagaku.49.961

Publishing type：Research paper (scientific journal)  

One simple example of a rapid flow-through analysis (RFA) system based on ion-selective optode detection was demonstrated, and its performance was discussed. The RFA is a flow analytical method based on a reagentless analyte flow injection system combined with an analyte selective monitor/sensor such as an optical chemical sensor (optode) involving mathematical response simulation using a semi-empirical theoretical response equation. The response of RFA with optode detection can basically be expressed by the equation derived from Fick's law of diffusion and Kubelka-Munk's law, and two other variable parameters in relation to the experimental response. Mathematically simulated results using the proposed equation indicated that the equation can be useful for the prediction of the flow analytical response. In the present demonstration of RFA, highly selective ionophores based on 16-crown-5 derivatives were used for the ion-sensing component of the optode for sodium ion determination. A sodium ion-selective optode based on C14-DD16C5 and KD-A3 exhibited high selectivity for sodium ion over potassium ion, with a selectivity coefficient (logK(i,j)(opt)) of -2.6. This optode responded to sodium ion in the concentration range from 10-5 to 10-1M at pH 8.0 by measuring the absorbance change at 510nm in the sensing phase of the optode. The RFA system equipped with a sodium ion-selective optode offers a reagent-less advanced ion-determination methodology for conventional flow injection analysis. Copyright (C) 1999 Elsevier Science B.V.

DOI： 10.1016/S0003-2670(99)00482-1

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

The preparation and response features of a micrometer-sized sodium ion- selective fiber optode based on a liquid membrane were described. The sensing membrane is a plasticized poly(vinyl chloride)-based copolymer with a neutral sodium ionophore and an anionic dye. To fabricate a micrometer-sized fiber optode, a 'micropipet fabrication method' was proposed to fix a liquid membrane-based optode on the small tip of an optical fiber probe, in which the optode size can be varied from ~1 μm to more than 10 μm. Our first optode incorporated a 16-crown-5 derivative as the neutral sodium ionophore and a dibromofluorescein derivative as the single-emission anionic dye. The sensor response was monitored by measuring the fluorescence intensity under a time-resolved photon-counting method. At the first stage of the investigation, it was found that the ionophore including a sodium ion in its cavity leached from the membrane phase. However, we have discovered that the problem can be resolved by using a 'tailed' ionophore, which is an ionophore possessing a lipophilic long alkyl chain. The 'tail' of the ionophore functions as an 'anchor' that prevents leaching of the ionophore from the membrane phase into the water phase. The anchor effect of the tailed ionophore was clearly demonstrated with 6-μm-sized sodium ion-selective optodes. Using a 3-μm-sized optode, the sensor response was examined and successfully explained by the response theory. The size limitation of the optode was also examined using the response features of a 1.5-μm-sized fiber optode. The second optode incorporated a tailed 16-crown-5 derivative as the neutral sodium ionophore and a coumarin derivative as the dual-emission anionic dye. The second optode is more practical than the first optode because the problem of fluorescence distortion due to photobleaching and solvent effect is resolved by ratiometric calibration where the sensor response is monitored by the spectral shift of the dual-emission fluorescence. The sensor response of an 8-μm-sized fiber optode having ratiometric calibration was examined and successfully explained by the response theory.

DOI： 10.1021/ac981206+

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

The chemistry of the ion-selective optode has recently been recognized as a highly selective ion-sensing methodology which involves a highly selective complexation between an ionophore and a primary ion. This simple methodology allowed the construction of several kinds of ion-sensing devices which enabled highly selective ion determination for analytical purposes. In this review, sensing devices are divided into the following four types with different analytical uses: (1) flow analytical devices (flow-through optode); (2) waveguide devices (waveguide optode); (3) sensing plate devices (sensing plate optode, film optode); (4) fiber-optic devices (fiber optode). The characteristics of each device are explained and discussed with experimental data, and the future prospects for ion-selective optode methodology are described. Copyright (C) 1999 Elsevier Science B.V.

DOI： 10.1016/S0165-9936(99)00137-5

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

By utilizing "multiinformation dyes (MIDs)", which have plural spectral change characteristics such as an absorption maximum wavelength (λmax) shift based on a polarity change and an absorbance change due to protonation, novel λmax-tunable ion-selective optodes were proposed and prepared by employing MIDs with membrane solvents having different polarities. For controlling the detecting λmax of the optode, the novel polar membrane solvent [2-[[6-(2-nita)phenoxy)hexyl]oxy]methyl]isobutane-1,3-diol was designed and synthesized, which was used together with a typical membrane solvent nitrophenyl octyl ether. By mixing these two membrane solvents, the λmax position of the optode detection wavelength can be shifted and controlled and was successfully applied to a λmax-tunable Li+-selective optode based on a highly Li+-selective ionophore TTD14C4. The λmax tuning technique is useful for preparing an optode system using a low-cost light source such as a light-emitting diode or a popular laser. © 1998 American Chemical Society.

DOI： 10.1021/ac980757x

Publishing type：Research paper (scientific journal)  

The molecular design of multi-functional dyes, called 'multi-information dyes' (MIDs), and their application to several methods of optical chemical sensing were proposed. The concepts for the molecular design of the dyes were as follows: (1) the introduction of multiple (both positive and negative) charges on both ends of the large conjugated π-electron system of the dye molecule, so that the dye interacts with many chemical species or environments; (2) the introduction of different substitution (electron-donating or electron-accepting) groups in the conjugated π-electron system of the dye molecule, so that the dye has a different pK(a) and solvatochromic property; and (3) the introduction of an immobilization site in the dye molecule, so that the dye can be easily prepared as a sensing probe.Based on these concepts, sixteen kinds of dyes were synthesized, all being of the merocyanine type. The basic color-change characteristics of the MIDs were evaluated with a homogeneous system and a solvent extraction two-phase system. Some of these dyes (e.g., KD-M5) offered two-dimensional sensing information for sensing the water content in organic solvents, in which the absorbance maximum wavelength (λ(max)) shift and absorbance change were both utilized as the detecting signals.From these results, the MIDs developed in this study have several possibilities for analytical applications in optical chemical sensing such as pH sensing, ion sensing, neutral molecule sensing, and water content-sensing in organic solvents. Copyright (C) 1998 Elsevier Science B.V.

DOI： 10.1016/S0003-2670(98)00421-8

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

In this paper, an active PVC optical thin-film waveguide sensor is proposed which can select a specific ion and measure its concentration. The waveguide was fabricated using the plasticized poly(vinyl chloride) (PVC), bis ethyl hexyl phthalate (BEHP), lipophilic anionic dye (LAD-3), and ionophores (K23E1 for calcium, or C14DD16C5 for sodium) in which the PVC membrane plays important roles for both incident light propagation and selective ion sensing. In order to investigate the applicability of this sensor to Ca2+ and Na+ measurement, the sensor was applied to sample solutions whose CaCl2 concentration was gradually altered. Also, we applied it to a NaCl solution. In this experiment, we found that this PVC thin-film waveguide possessed good light propagation for argon laser light at 488 nm. Also, the comparison of the experimental responses between the active waveguide sensor and a commonly utilized waveguide sensor (evanescent waveguide spectrometry), indicated that the sensitivity of the active waveguide sensor is obviously higher than that of the conventional waveguide sensor. These findings suggested that the proposed PVC waveguide ion sensor is applicable to the determination of various ions with high sensitivity.

DOI： 10.1016/S0003-2670(97)00006-8

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Rapid flow-through analysis (RFA) based on optode (optical chemical sensor) detection is proposed, and its performance is discussed using the RFA system equipped with a pH-sensitive optode as a model case. To demonstrate and understand the usefulness of the RFA system, long-lifetime pH-sensitive optodes were prepared using a hydrophilic poly(hydroxyethyl methacrylate) (poly-HEMA) membrane covalently immobilized with a dye containing an amino group such as Congo Red or Nile Blue. The response equation for the optode with the RFA system was proposed, and satisfactory estimation for rapid pH analysis was obtained. The proposed RFA can stand as an advanced method for conventional flow injection analysis.

DOI： 10.1021/ac951149+

Kind of work：Joint Work  

Kind of work：Joint Work  

DOI： 10.1295/kobunshi.45.100

CiNii Article

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Several N-hydroxylamide derivatives were developed, exhibiting high selectivity for Hg2+, which are useful as sensory molecules for a potentiometric ion sensor.

DOI： 10.1246/cl.1996.1011

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Highly selective ion sensing film optodes for the determination of four kinds of ions (Na+, K+, Ca2+, Cl-) were prepared with plasticized PVC membranes containing the respective ionophores and a newly prepared lipophilic anionic dye. The principle of the optical response of these sensing films is based on ion-pair extraction of the ion-ionophore complex and a deprotonated or protonated lipophilic anionic dye. These film optodes are for disposable use and can detect ions by simply immersing them into a glass vessel which is filled with the samples solution and determining the ion by measuring the absorbance change at the respective maximum absorption wavelength of the lipophilic anionic dyes in the visible region. The theoretical response of these films was also discussed based on the ion-extraction model. The ion selectivity of these films was sufficient for the determination of Na+, K+, Ca2+ and Cl- in diluted serum samples, and satisfactory results were obtained in serum analysis. © 1995.

DOI： 10.1016/0925-4005(95)01711-9

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

© 17CBMS-0001. In this study, we developed a novel microfluidic platform combining reagent-release capillaries and hydrogels (RRCs and RRGs) for multiple enzyme activity assay. Developed RRC-assembled device provided a rapid, simple and multiple enzyme activity assay by only introducing a sample solution by capillary action. The enzyme reaction products were then concentrated on the basis of double sweeping using both the cationic and anionic micelles from the RRGs by applying voltage. As a result, a rapid, simple, sensitive, and simultaneous assay of caspase-3, trypsin, and alkaline phosphatase was successfully realized by the developed device.

© 17CBMS-0001. In this study, novel concept of micrometer-scale spectrometer (P-spectrometer) composed of imprinted photonic crystal nanocavities (PCNs) was proposed. The P-spectrometer has sufficient size to integrate into microfluidic devices and possible to detect light absorption high sensitively. Here we fabricated a PCN as unit cell of P-spectrometer using nanoimprint lithography, and experimentally confirmed for the first time that the fabricated PCN worked as a detection module for 560 nm wavelength.

© 17CBMS-0001. This paper reports the preparation of the polyethylene glycol-coated graphene oxide (PEG-GO) possessing highly dispersive, fluorescence quenching and size separation functions, and its application to “single-step” immunoassay. The present immunoassay microdevice was fabricated by combining PDMS microchannel arrays separately immobilizing PEG-GO and fluorescently-labelled antibody as soluble coatings. When a sample solution was introduced into the microdevice by capillary action, all the reagents spontaneously mixed, reacted, and free fluorescently-labelled antibody was penetrated into the PEG structure and effectively quenched. Thus the antigen was successfully detected by single-step operation from the change of fluorescence intensity.

© 17CBMS-0001. In this work, we synthesized a new material, “dyed plasticizer”, [P66614]2[NP] which consists of two trihexyltetradecylphospohonium cations and a naphtholphthalein anion, and prepared the plasticized PVC membrane. We systematically investigated the reversibility of response, sensitivity and response time, and demonstrated rapid and highly-sensitive anion sensing based on the coextraction of anions and protons into the bulk of the plasticized PVC membrane. We prepared a microchannel array device combined with plasticized PVC membrane, and Cl- sensing using flat-bed scanner was successfully demonstrated.

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. A plasticized PVC sensor membrane using “dyed plasticizer”[1], where exceptionally high concentration of dye was retained, exhibits rapid and highly-sensitive color change. In this study, we applied a PVC membrane based on “dyed plasticizer” to heparin detection, and prepared the microchannel array integrated with the plasticized PVC membrane. The results presented here demonstrated that our developed micro analytical device was proved to be effective for rapid and highly-sensitive measurement of heparin.

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. In this work, we developed a PDMS-based microdevice capable of highly sensitive and rapid calcium ion quantitation based on poly(vinylchloride) (PVC) membrane containing newly-developed calcium-selective “dyed plasticizer”. First, we demonstrated rapid and highly-sensitive cation sensing based on cation-proton exchange at aqueous/membrane interface. Finally, we fabricated a PDMS microchip using this new material for the single step and simultaneous data collection of Ca2+ calibration data, and preliminary results for Ca2+ calibration was obtained in the range of 10-4 ~ 10-1M.

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. Digital electrophoresis microdevices consisting of convex-concave-type cartridges (CCCs) were developed for a multi-dimensional fractionation of proteins. The various CCCs containing a different functionalized hydrogel allow simple connection and detachment of each other, which enables multi-dimensional separation, preconcentration, and fractionation of proteins based on digital electrophoresis using the serially connected hydrogels. As a typical result, two-dimensional fractionation of model proteins based on isoelectric focusing and molecular sieving was successfully achieved by the developed microdevices.

J-GLOBAL

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. Here, we report the newly prepared polyethylene glycol (PEG)-coated graphene nanoparticle (PGN) possessing fluorescence quenching and size separation functions, and its application to single-step immunoassay. The PGN was prepared by the coacervation method, and by optimizing the preparation condition, PGN successfully separated the free fluorescently-labelled antibody and immunocomplex by size difference. By separately adsorbing fluorescently-labelled antibody and PGN onto the inner wall of two PDMS microchannel arrays and combining, the present immunoassay microdevice was prepared. Finally, by introducing a sample solution by capillary action, we successfully measured the C-reactive protein (CRP) as a model antigen within 2 min.

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. Herein, a novel method for the intact immobilization of a very thin and soft PVC membrane on a convex-shaped PDMS surface is described. The present method using PET film mask and PVA film as a sacrificial layer allowed successful immobilization of an intact PVC membrane using an ionic liquid-based dye only on the convex-shaped PDMS surface. The present method is applicable for various shapes of PDMS structure without any deformation or increase of the inhomogeneity of PVC membrane.

CiNii Article

CiNii Article

CiNii Article

CiNii Article

Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

© The Japan Society for Analytical Chemistry. In this study, a fast and easy-to-use capillary-type microdevice for competitive bioassay is proposed. The device is composed of polydimethylsiloxane (PDMS) microchannel arrays that separately immobilize polyethylene glycol (PEG) coating, which contained graphene oxide (GO)-analyte conjugates and fluorescently-labelled receptor proteins. The working principle of the device involved the spontaneous dissolution of the PEG coating, subsequent mixing and reaction with analyte to give fluorescence response, triggered by the capillary action-mediated introduction of a sample solution. For principle verification, a competitive biotin assay was successfully demonstrated within 20 s in a single-step operation by detecting the change in fluorescence via microscopy.

DOI： 10.2116/analsci.33.969

PubMed

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

© 2015 The Japan Society of Applied Physics. In this study, for medical diagnostics, the polymer-based two-dimensional photonic crystal (2D-PhC) was fabricated using nanoimprint lithography (NIL)-based nanooptical device fabrication technology that called as "printable photonics". In addition, polymer-based 2D-PhC was applied for detection of fibrinogen which is a biomarker to diagnose the chronic obstructive pulmonary disease (COPD) using antigen-antibody reaction. Based on printable photonics technology, the polymer-based nanooptical device which act in visible region could be fabricated cost effectively and easily. Furthermore, by using our polymer-based 2D-PhC, fibrinogen could detect sensitively and rapidly.

DOI： 10.1109/MOC.2015.7416439

To realize a simple, highly sensitive, and "designable" assay of proteins, a novel digital electro-phoresis device is developed by connecting capillary cartridges filled with a different "functionalized hydrogel". The multi-step digital separation based on different electrophoresis modes could be easily demonstrated by designing, arranging, and connecting the capillary cartridges without any complicat-ed fabrications. The model proteins were easily concentrated and separated on the basis of digital molecular sieving electrophoresis by using the developed device, indicating that desirable assays could be realized by the proposed concept.

This paper reports the preparation and characterization of graphene oxide (GO)-containing hydrogel possessing fluorescence quenching and size separation functions, and its application to single-step immunoassay. Immunoassay microdevice was fabricated by combining PDMS microchannel and GO-containing hydrogel prepared by 2-hydroxyethyl methacrylate (HEMA). Free fluorescently-labeled antibody and immunocomplex was successfully separated by size difference and the former was selectively penetrated into hydrogel to directly adsorb onto bare GO surface for fluorescence quenching-based immunoassay. Here, the concentration of model antigen (human IgG) was successfully measured.

J-GLOBAL

© 15CBMS-0001. This paper reports a newly proposed online sample concentration method named "double sweeping" using hydrogels containing mixed micelles with cationic or anionic charges. Highly effective online concentration of fluorescent molecules typically employed in enzyme activity assays was achieved by the proposed method using the mixed micelles, which overcame the sensitivity problems in previous microdevices and provided up to 200-fold enhancement of the obtained signal. The application of the developed device to the enzyme activity assay was successfully achieved, resulting in the highly sensitive enzyme activity assay with small consumptions of the sample and reagents.

© 15CBMS-0001. A microfluidic isoelectric focusing (IEF) device with reagent-release hydrogels (RRGs) were newly developed for a highly sensitive enzyme assay using a two-steps enzyme reaction of bifunctional substrates. Problems in previous assays such as low sensitivity and a difficulty in the separation of an intermediate and a final product of the reaction were solved by the developed IEF device, providing the accurate kinetic assay of the two-steps enzyme reaction with high sensitivity and a simple experimental procedure.

© 15CBMS-0001. This paper reports a rapid, sensitive, and simple enzyme activity assay device by the combination of electrophoretic filtration of enzymes and detection using a hydrogel immobilizing fluorescent substrates (detection gel). The detection gel and a dense hydrogel to filtrate enzymes were successively prepared in a capillary. The effective filtration of low-concentration enzymes using the dense hydrogel accelerated the enzyme reaction of the fluorescent substrate in the detection gel, providing the rapid response and the enhancement of the fluorescence intensity.

© 15CBMS-0001. This paper reports the development of a rapid and highly sensitive electrophoretic immunoassay device using a hydrogel immobilizing fluorescent substrates. Electrophoretic filtration of immunocomplex using a hydrogel allowed the bound/free separation and sensitive detection. Enzyme reaction of the immobilized substrate was accelerated by electrophoretic filtration of the enzyme-labeled antibody, providing the rapid and sensitive detection. The proposed device using the hydrogel immobilizing fluorescent substrates overcame the problems in conventional immunoassays such as the long analysis time, low sensitivity, and complex procedure.

© 15CBMS-0001. Here, we report newly fabricated mass-producible capillary-assembled microchip (CAs-CHIP) for multiple bioanalytical sensing with single-step operation that is expected to be utilized in the field of point of care testing (POCT). Today, there are huge number of studies on Micro-Total Analysis Systems toward medical diagnosis, however the usability and portability are still the problems. To improve these, we developed the new CAs-CHIP that is mainly composed of square glass capillaries possessing chemical sensor functions arrayed side-by-side. The integration of various capillary-type sensors would allow us to develop the universal and on-demand microdevices for use in POCT.

Publishing type：Research paper, summary (international conference)  

© 14CBMS. This paper reports an evaluation of a separation and online concentration of proteins based on molecular sieving and affinity trapping using stacked functional hydrogels prepared within capillaries towards a development of a novel microfluidic western blotting device based on multi-dimensional digital electrophoresis using a capillary-assembled microchip (CAs-CHIP). To realize the rapid, sensitive, and selective multi-detection of proteins, various capillaries filled with a different functionalized hydrogels were prepared. The prepared capillaries successfully provided the separation and up to 180-fold concentration due to a molecular sieving effect, or selective capturing using antibodies.

© 14CBMS. In this study, hydrogel-based two-dimensional photonic crystal (2D-PhC) including acrylic acid for biosensing application was successfully developed by using nanoimprint lithography (NIL). This hydrogel-based 2D-PhC enables to reduce non-specific adsorption of proteins and to simply immobilize biorecognition elements such as an antibody onto the 2D-PhC surface via a carboxyl group of acrylic acid. By using this hydrogel-based 2D-PhC, refractive index change was detected as a reflection intensity change, and the detection of antigen-antibody reaction could be achieved.

© 14CBMS. Here, we report ultrafast and single-step immunoassay within 30 seconds using functional graphene oxide and chemically-modified capillary-type microdevice. Today, there are huge number of studies on bioassay using graphene oxide (GO) which has exceptional fluorescence quenching ability, however there are some problems that are the needs for step-by-step operation and long analysis time depending on the reaction scale. We solved such problems by using capillary-type microdevice immobilizing polyethylene glycol (PEG) coating inside capillary, which contains antibody-conjugated GO and fluorescently-labeled antibody. By using the present device, simply introducing a sample solution by capillary action allowed successful single-step immunoassay.

© 14CBMS. We developed two-dimensional digital electrophoresis using a mosaic-like multi-layered hydrogel plate (mosaic hydrogel). In two-dimensional digital electrophoresis, proteins are separated and concentrated nearby the interface of the layered hydrogels on the basis of IEF and molecular sieving by functionalized mosaic hydrogel. The mosaic hydrogels was prepared by photolithographic technique, and it was confirmed that the prepared hydrogel enables a discontinuous separation and on-line concentration of proteins simultaneously. Two-dimensional digital electrophoresis of proteins based on isoelectric focusing and molecular sieving were successfully demonstrated using the developed device integrating the mosaic hydrogel.

© 14CBMS. This paper reports the development of a rapid, highly sensitive and simple electrophoretic immunoassay device using a hydrogel immobilizing fluorescent substrate. To achieve the proposed electrophoretic immunoassay, electrophoretic behavior of enzyme-labeled antibody and subsequent reaction with fluorescent substrate immobilized within hydrogel were investigated. Especially, it was confirmed that a novel detection scheme based on an on-line concentration of enzyme-labeled antibody using the hydrogel could accelerate the enzyme reaction, providing a rapid and highly sensitive detection with a simple procedure.

© 14CBMS. Here, we report the development of functional hydrogel array on a flexible polymer sheet, allowing fabrication of mass-producible and single-step multi-sensing device, which can be used for the discrimination of sick patient serum among many serum samples. In the present study, we designed and synthesized a fluorescent substrate monomer for hydrogen peroxide (H<inf>2</inf>O<inf>2</inf>) which was immobilizable to hydrogel by copolymerization, and preliminary investigation for the development of single-step multi-sensing device for glucose, cholesterol, and uric acid, using this monomer was carried out.

© 14CBMS. In this study, we developed polymer/TiO2 hybrid two-dimensional photonic crystal (2D-PhC) optical sensor that has the ability of fluorescence enhancement based on the matching of fluorescence wavelength and confineable wavelength of 2D-PhC for the first time. Ten and three times fluorescence enhancement were achieved for PMMA/TiO2 and plasticized PVC/TiO2 2D-PhCs, respectively.

© 14CBMS. In this study, a polymer-based 2-dimensional photonic crystal (PhC) cavity for highly sensitive optical sensor was successfully designed and fabricated. By using this sensor, label-free, highly sensitive, and cost effective biosensor can be realized. We demonstrated introduction of a cavity into polymer-based PhC to confine the visible light (650 nm) into the cavity by the photonic bandgap (PBG) and found that the present device possess single polymer layer detection sensitivity in Layer-by-Layer (LbL) deposition process.

The urokinase (uPA) activity is widely recognized as a potential parameter for diagnosis of breast cancer metastasis. In this study, we developed novel sensing mechanism using peptides-immobilized two dimentional photonic crystal (PhC) for determination of uPA activity. The detection limit of developed device was 80 mIU/mL, and this value is lower than diagnosis concentration of breast cancer metastasis[1].

A conventional flatbed scanner was employed for direct detection of colored solution in capillary-based micro analytical device. Since the flatbed scanner is a common and inexpensive detection equipment (usually ∼ 50 USD), establishing the method for detecting colored solution within capillary or microchannel would allow us widespread use of microfluidic devices including point-of-care (POC) testing. Here, scan conditions (resolution, scan direction) were systematically investigated, and determined detection limits of various colored solutions within 100 jam-sized square capillary. Finally, pH sensing was performed as a demonstration and successfully obtained a calibration curve. Copyright © (2013) by the Chemical and Biological Microsystems Society All rights reserved.

We fabricated hydrogel-based photonic crystals (PhC) using poly(ethyleneglycol) diacrylate (PEGDA-PhC) which included with tungsten trioxide (WO3) for refractive index sensor applications. Reduction of nonspecific adsorption by using PEGDA as base material and sensitivity enhancement by adding WO3particle were successfully achieved. Moreover, simple functionalization by amino group using allylamine during polymerization was investigated.

In this study, we developed plasticized poly (vinyl chloride) (PVC) based two-dimensional photonic crystal (2D-PhC) optical sensor for the first time, that has the ability of ion selective extraction. Plasticized PVC is well known as an optical ion sensor material[1]. This novel plasticized PVC based 2D-PhC was fabricated by nanoimprint lithography (NIL). Pattern transfer was confirmed using scanning electron microscopy (SEM). The reflection peak of PVC-based PhC could be observed at 580 nm by diffraction of light. Potassium sensing selective elementswere dissolved in plasticized PVC-based PhC, and successfully observed the K+-selective response by the reflection peak intensity change.

Here, we report a new principle of single-step enzyme immunoassay by using a newly-synthesized lipophilic fluorescent substrate, which can be immobilized to a plasticized polyvinyl chloride (PVC) coating by hydrophobic interaction. The plasticized PVC was coated inside a square glass capillary and further coated by hydrogel coating as a second layer which worked for discriminating antigen-antibody complex and free antibody. Then soluble coating containing enzyme-labeled antibody was coated as a third layer to successfully prepare a capillary for single-step immunoassay. Introducing a sample solution by capillary action allowed successful single-step immunoassay of cancer marker protein, alphafeto protein (AFP). Copyright © (2013) by the Chemical and Biological Microsystems Society All rights reserved. All rights reserved.

Caspase-3 inhibitor assay important in drug development was successfully integrated into "Single-Step" by solving the problem of low-activity enzyme immobilization. We developed a combinable PDMS capillary (CPC) sensor for enzyme inhibition assay using the concave- and convex-type PDMS substrates. In the conventional enzyme inhibitor assay, complicated analytical procedures, such as mixing and dissolution, are required. In contrast, this sensor allows single-step assay possible only by sample introduction via capillary action. Total reaction time was reduced from lh to 3-5 min, and simultaneous acquisition of multiple data required to determine IC50 value was successfully achieved by using CPC sensor array.

Simple and rapid immunoassay based on micro isoelectric focusing (IEF) was newly developed by using hydrogels containing acid or base as ion suppliers. The prepared hydrogels have enough mechanical strength and can eliminate a handling operation of acid and base solutions which often causes an unfavorable hydrodynamic flow in conventional IEF. As a result, the proposed device provided the easy-to-use reagent release capillary-IEF (RRC-IEF). The RRC-IEF with reagent release hydrogels was also applied to microscale immunoassay, realizing a simple and rapid immunoassay by concentration and separation of the bound/free analytes within 10 min.

This paper reports the development of simple and highly-sensitive enzyme assay microdevice by the combination of reagent-release hydrogel (RRG) and reagent-release capillary (RRC). On-line concentration of fluorescent enzyme reaction products and change of pH condition in RRC were demonstrated by using RRGs containing micellar or basic solution, providing highly-sensitive assay of enzyme activity without a problem of pH mismatching. Copyright © (2013) by the Chemical and Biological Microsystems Society All rights reserved. All rights reserved.

Sensitivity enhancement of enzyme-linked immunosorbent assay (ELISA) in capillary-based microdevice was achieved by the concentration of fluorescent final enzyme reaction product using capillary-isoelectric focusing (CIEF). In order to realize this, fluorescent substrate molecule, which can change its structure into ampholyte ion after an enzyme reaction, was newly synthesized and used. Approximately seven-fold enhancement of sensitivity and 1-2 orders of magnitude lower the detection limit were achieved.

This paper reports a novel microdevice showcasing the rapid and simultaneous multi-analyte sensing of different serum components; glucose, cholesterol, pH and alkaline phosphatase (ALP). Discrimination between normal and high analyte concentration in a serum sample was achieved and results were obtained within minutes after sample introduction. The complexity of microdevice fabrication and fluid handling from our previous system, capillary-assembled microchip (CAs-CHIP) [1-2], has been circumvented in this work that led to a simple fabrication and easy operation. Furthermore, we have exploited the fluorescein-based probes for simultaneous multi-analyte sensing by using only one fluorescence filter.

DOI： 10.2116/analsci.28.1

DOI： 10.2116/analsci.27.349

PubMed

In this study, we fabricated the Combinable-PDMS Capillary (CPC) sensor allowing single step assay by simply introducing a sample solution by capillary force. CPC is composed of a convex-shaped PDMS plate immobilizing undissolvable reagents and a concave-shaped PDMS plate immobilizing dissolvable reagents. Since the concave-shaped PDMS has a deeper channel size compared to that of convex structure, combining these PDMS plates allowed easy fabrication of capillary structure possessing different reagents inside the same capillary. This method is useful for immobilizing two different reagents which reacts each other. As an application, we carried out a single-step enzyme inhibitor assay using the CPC. First, a fluorescent substrate is immobilized with Polyethylene glycol (PEG) on concave-shaped PDMS plate. Secondly, trypsin, an enzyme, is quantitatively immobilized using Layer-by-Layer (LbL) technique on convex-shaped PDMS plate. Then these PDMS plates are combined to fabricate CPC for enzyme inhibitor assay. Buffer solution containing various concentrations of leupeptin, a trypsin inhibitor, is introduced into the capillary, then inhibition curve was obtained. © 2009 CBMS.

CiNii Article

Publishing type：Book review, literature introduction, etc.  

This review accounts for the current development in microfluidic immunosensing chips. The basic knowledge of immunoassay in relation to its microfluidic material substrate, fluid handling and detection mode are briefly discussed. Here, we mainly focused on the surface modification, antibody immobilization, detection, signal enhancement and multiple analyte sensing. Some of the clinically important currently implemented on the microfluidic immunoassay chips are C-reactive protein (CRP), prostate specific antigen (PSA), ferritin, vascular endothelial growth factor (VEGF), myoglobin (Myo), cardiac troponin T (cTnT), cardiac troponin I (cTnI), and creatine kinase-cardiac muscle isoform (CK-MB). The emerging microfludic immunosensor technology may be a promising prospect that can propel the improvement of clinical and medical diagnosis. © 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aca.2008.01.064

PubMed

A novel capillary immunosensor capable of single-step heterogeneous sandwich immunoassay for capillary-assembled microchip (CAs-CHIP) integration is presented. It is mainly composed of a covalently attached primary antibody and non-covalently immobilized secondary labeled antibody in the inner wall of the square glass capillary. Sample solution containing antigen enters via capillary force resulting to the release of the secondary labeled antibody and immunocomplex formation occurs on the capillary wall followed by fluorescence detection. Here, the capillary immunosensor contains the fluorescein-labeled and unlabeled anti-human IgG for non-covalent and covalent immobilization, respectively. The immunosensor respond successfully to changes in human IgG concentration. © 2008 CBMS.

CiNii Article

© 2007 CBMS. A simple sensing conduit for capillary-assembled microchip (CAs-CHIP) integration that is capable of metabolite detection by fluorescence microscopy is developed. This sensing capillary called enzyme-release capillary (ERC) contains enzyme/s that are adsorbed inside the square glass capillary with the aid of polyethylene glycol acting as scaffold. Metabolite solution enters the conduit via capillary force and the enzyme/s are released leading to the detection of a fluorescent biochemical product. Here, the stability of the E-galactosidase immobilized capillary is observed to last for almost a month. Moreover, the co-immobilized glucose oxidase-peroxidase-thiamine system for glucose measurement showed a detection limit of about 30 PM.

© 2007 CBMS. The basic concept of “drop-and-sip” fluid delivery on a reagent-release capillary-based capillary-assembled microchip (RRC-based CAs-CHIP) has been published elsewhere [1]. Here, we intend to present significant details on the active and passive microfluidics present in the RRC-based CAs-CHIP. More importantly, this fluid handling technique is implemented on a real sample, HeLa cell lysate, wherein chemotheraphy-induced cell death with a certain drug, doxorubicin, is profiled with ten different protease-sensing capillaries embedded on the CAs-CHIP comparing the enzyme activities in a doxorubicin-treated and untreated cell lysates simultaneously.

CiNii Article

CiNii Article

A novel concept of assembling various chemical functions onto a single microfluidic device is proposed. The concept, "CAPILLARY-ASSEMBLED MICROCHIP (CAs-CHIP), involves embedding chemically functionalized square capillaries onto a lattice microchannel network fabricated on polydimethyl siloxane (PDMS) possessing same channel dimensions to outer dimensions of square capillaries (Figure 1). This approach would allow easy fabrication of chemically-functionalized microfluidic device by freely embedding functional square capillaries on PDMS microchannel network. Here we report a fabrication method of CAs-CHIP, and an application for preparing a multiple chemical sensing chip as an example.

In this study, focus has been placed on potentiometric measurement of ammonia ions for BUN (blood urea nitrogen) and Creatine sensors by using new 19-membered crown ionophore, TD19C6 (3wt%), PVC (30wt%) membranes along with anionic additives (10mol %) K-TCPB and TFPB, and plasticizers (67wt%) BBPA and TOTM. The screen printed electrodes Ag/AgCl (250mm in diameter), newly developed non-liquid junction reference electrode, painless needle and disposable polycarbonate chip (PC), designed of using a trace of amount of whole blood extracted from painless needle is demonstrated. Copyright © 2005 by the Transducer Research Foundation, Inc.

Publishing type：Book review, literature introduction, etc.  

This review describes our recent research on miniaturization of chemical systems. We have developed a miniaturization methodology based on pressure-driven multiphase laminar flow and a highly sensitive detection tool, the thermal lens microscope. Some representative applications of the methodology in the fields of analysis, synthesis, and bioassay are described.

DOI： 10.1002/elps.200305661

PubMed

CiNii Article

Publishing type：Book review, literature introduction, etc.  

This review focuses on chemical and biochemical analysis systems using pressure-driven microfluidic devices or microchips. Liquid microspace in a microchip has several characteristic features, for example, short diffusion distances, high specific interfacial area and small heat capacity. These characteristics are the key to controlling micro unit operations and constructing new integrated chemical systems. By combining multiphase laminar flow and the micro unit operations, such as mixing, reaction, extraction and separation, continuous flow chemical processing systems are realized in the microchip format. By applying these concepts, several different analysis systems were successfully integrated on a microchip. In this paper, we introduce the microchip-based chemical systems for wet analysis of cobalt ion, multi-ion sensors, immunoassay, and cellular analysis. © 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0169-409X(02)00225-9

PubMed

Publishing type：Book review, literature introduction, etc.  

This review focuses on the integration of chemical and biochemical analysis systems into glass microchips for general use. By combining multiphase laminar flow driven by pressure and micro unit operations, such as mixing, reaction, extraction and separation, continuous-flow chemical processing systems can be realized in the microchip format, while the application of electrophoresis-based chip technology is limited. The performances of several analysis systems were greatly improved by microchip integration because of some characteristics of microspace, i.e., a large specific interface area, a short molecular diffusion time, a small heat capacity and so on. By applying these concepts, several different analysis systems, i.e., wet analysis of cobalt ion, multi-ion sensor, immunoassay, and cellular analysis, were successfully integrated on a microchip. These microchip technologies are promising for meeting the future demands of high-throughput chemical processing.

DOI： 10.2116/analsci.19.15

PubMed

CiNii Article

CiNii Article

CiNii Article

CiNii Article

CiNii Article

CiNii Article

In this paper, a PVC optical thin-film waveguide sensor is proposed which can select a specific ion and measure its concentration depending on materials added into waveguide membrane. In order to investigate the applicability of this sensor to Ca++ and Na+ measurement, the waveguide was fabricated with poly vinyl chloride (PVC), bis ethyl hexyl phthalate (BEHP), lipophilic anionic dye (LAD-3), and ionophores (K23E1 for Ca++ or C14DD16C5 for Na+). For this purpose, we applied the sensor to sample solution whose CaCl2 concentration was gradually altered. Also, we applied it to NaCl solution. In this experiment, we found that this PVC thin film waveguide possessed good ray propagation at 488 nm of argon laser. In addition, it showed the chemical reaction to ion enough to select it and measure its concentration. These findings suggested that the proposed PVC waveguide ion sensor is applicable to various ions.

DOI： 10.1295/kobunshi.45.100

CiNii Article

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Oral presentation (general)  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Oral presentation (general)  

Presentation type：Poster presentation  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (invited, special)  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (general)  

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (general)  

Presentation type：Poster presentation  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (general)  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Oral presentation (invited, special)  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

11/1

FDセミナー「反転授業実践セミナー」
日時：2021年 3月15日（月）14：00～16：30
　　　【第1部】14：00～15：00
　　　　講師　森澤正之氏（山梨大学 大学院総合研究部 工学域　教授）
　　　【第2部】15：30～16：30
　　　　講師　鹿住大助氏（島根大学 教育・学生支援機構 大学教育センター　准教授）

2020年度第2回工学全体FDセミナー
「研究室指導に必要なコーチング技能入門WS
－学生が自己効力感を高める工夫－」

日時：9月7日（月）13:30-15:30
場所：Zoomによるオンライン開催　

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review 

Type：Scientific advice/Review