Amperometric biosensor based on reductive H2O2 detection using pentacyanoferrate-bound polymer for creatinine determination.

Pentacyanoferrate-bound poly(1-vinylimidazole) (PVI[Fe(CN)5]) was selected as a mediator for amperometric creatinine determination based on the reductive H2O2 detection. Creatinine amidohydrolase (CNH), creatine amidohydrolase (CRH), sarcosine oxidase (SOD), peroxidase (POD), and PVI[Fe(CN)5] were crosslinked with poly(ethylene glycol) diglycidyl ether (PEGDGE) on a glassy carbon (GC) electrode for a creatinine biosensor fabrication. Reduction current was monitored at −0.1 V in the presence of creatinine and O2. It is revealed that PVI[Fe(CN)5] is suitable as a mediator for a bioelectrocatalytic reaction of POD, since PVI[Fe(CN)5] neither reacts with reactants nor works as an electron acceptor of SOD. The amounts of PVI[Fe(CN)5], PEGDGE, and enzymes were optimized toward creatinine detection. Nafion as a protecting film successfully prevented the enzyme layer from interferences. The detection limit and linear range in creatinine determination were 12 μM and 12–500 μM (R[2]= 0.993), respectively, and the sensitivity was 11 mA cm[−2] M[−1], which is applicable for urine creatinine tests. The results of the creatinine determination for four urine samples measured with this proposed method were compared with Jaffe method, and a good correlation was obtained between the results

ペンタシアノ鉄錯体ポリマーを用いた酸化酵素/ペルオキシダーゼ型バイオセンサに関する研究

京都大学0048新制・課程博士博士(農学)甲第17895号農博第2018号新制||農||1017(附属図書館)学位論文||H25||N4791(農学部図書室)30715京都大学大学院農学研究科応用生命科学専攻(主査)教授 加納 健司, 教授 三芳 秀人, 教授 小川 順学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Electrostatic and steric interaction between redox polymers and some flavoenzymes in mediated bioelectrocatalysis

H2O2-generating oxidase/peroxidase (POD)-based mediated biosensors are very useful to minimize interference, but require suitable mediators which work well only for POD but not against the oxidase. Pentacyanoferrate-bound poly(1-vinylimidazole) (PVI[Fe(CN)5]), PVI[Os(dcbbpy)2Cl] (dcbbpy = 4, 4′-dicarboxy-2, 2′-bipyridine) and PVI[Os(dmebpy)2Cl] (dmebpy = 4, 4′-dimethyl-2, 2′-bipyridine) have been utilized to investigate the interaction with four kinds of H2O2-generating oxidases: glucose oxidase, sarcosine oxidase, choline oxidase (ChOD) and lactate oxidase. The mediated bioelectrocatalytic activities of the redox polymers for the enzymes have been determined by cyclic voltammetry in the presence of the substrates. The highly negatively charged PVI[Fe(CN)5] shows practically no mediating activity against the four flavoenzymes, but strong one to POD. On the other hand, PVI[Os(dmebpy)2Cl] with neutral ligands shows a high activity for the oxidases except ChOD. The mediating activity of PVI[Os(dcbbpy)2Cl] with negatively charged ligands is much smaller than that of PVI[Os(dmebpy)2Cl]. These results reveal that electrostatic repulsion and steric hindrance are enhanced by using negatively charged polymers to realize minimum activity against the oxidases

Development of disposable biosensing strips for the quality control of economic agricultural products

本研究開發兩種拋棄式檢測試片應用於茶品與蝴蝶蘭的品質管理。茶品品管檢測試片係利用拋棄式網印碳電極搭配流動注射系統檢測茶湯的氧化還原電位，藉此評定茶品發酵度。此法可減少電極表面上多元酚類的吸附(CV<1.2%)且達到高通量檢測 (樣品檢測數量 >20 hr-1)。透過檢測不同發酵度之自製包種茶，顯示此法與高效能液相層析法呈現高度相關性。研究另一檢測試片為蘭花蕙蘭嵌紋病毒(CymMV)檢測用免疫層析試片。將多株抗體結合40nm金粒子，可利用肉眼觀察到抗原抗體結合之呈色變化。本研究中，利用肌紅蛋白固定於金奈米粒子，透過肌紅蛋白內之heme基來增強luminol- H2O2 之化學發光強度，藉此降低試片檢測極限。Two types of disposable sensing strips were developed for the quality control of two economic agricultural products, tea and moth orchids. The sensing strip for tea quality is a screen-printed carbon-paste disposable electrode that can be incorporated into a flow injection manifold to estimate tea fermentation degrees based on the redox potentials of tea infusions. The flow-injection analytical manner reduced the adsorption of polyphenols on the electrode surface, reproducible (CV<1.2%) and high-throughput analytical results (>20 hr-1) were obtained. For pouchong tea samples with different fermentation degrees, the data show a high correlation with those obtained by HPLC.he second one is an immunochromatographic strip for detecting the Cymbidium mosaic virus in moth orchids (phalaenopsis spp.). To visualize the immunochromatographic process, polyclonal antibodies against the coat protein of the virus were attached onto 40nm gold nanoparticles. In the present study, myoglobin was immobilized onto the gold nanoparticles to enhance the luminol-H2O2 chemiluminescence by the prosthetic heme group of myoglobin.誌謝 i文摘要 iibstract iiiable of Contents ivist of Figures viiist of Tables ixomenclature xhapter 1 Introduction 1hapter 2 Literature Review 3.1 Estimation of tea fermentation degree 3.1.1 Chromatographic determination of tea catechins 3.1.2 CE determination of tea catechins 8.1.3 Spectrographic methods 9.1.4 Electrochemical and other methods 9.1.5 Fouling problems on the electrode surface 11.2 Virus diagnosis in orchids 12.2.1 Immunoassay procedures 12.2.1.1 Agar gel double diffusion method 12.2.1.2 Enzyme-linked immunosorbent assay (ELISA) 12.2.1.3 Quartz crystal microbalance (QCM) immunosensors 15.2.1.4 Magnetoreduction assay (MRA) 15.2.2 Nucleic acids procedures 17.2.2.1 Polymerase chain reaction (PCR)-based methods 17.2.2.2 QCM-based DNA biosensors 20.2.3 Other analytical methods 20.2.4 Novel immunochromatographic strip for biodiagnosis 21.2.5 Properties and applications of AuNPs in biodiagnosis 23hapter 3 Materials and Methods 26.1 Estimation of tea fermentation degree 26.1.1 Chemicals 26.1.2 Preparation of buffers and reagents 26.1.3 Preparation of tea infusions 26.1.4 Flow-injection manifold 27.1.5 Working electrodes 29.1.6 Manufacture of tea samples 29.1.7 HPLC estimation of tea catehins 31.2 Detection of CymMV and ORSV by a immunochromatographic strip 32.2.1 Chemicals 32.2.2 Preparation of reagents 32.2.3 Preparation of gold colloid 33.2.4 Conjugation of catalyst-AuNPs 34.2.4.1 Preparation of HRP-AuNPs 34.2.4.2 Preparation of Mb-AuNPs 34.2.4.3 Preparation of Mb-S-AuNPs 34.2.5 CL measurement of Mb-AuNP conjugates 37.2.6 Preparation of anti-CymMV IgG&Mb-AuNP conjugates 37.2.7 Immunochromatographic strip test 37hapter 4 Results and Discussion 39.1 Estimation of tea fermentation degree 39.1.1 Reliability of FIA system 39.1.2 Comparison of different working electrodes on sensor response 40.1.3 Redox potential measurement of black tea/green tea mixtures 41.1.4 Effect of indoor withering on redox potential and depletion of tea catechins 45.1.5 Measurements of commercial tea 49.2 Detection of CymMV and ORSV by an immunochromatographic strip 50.2.1 Synthesis of AuNPs 50.2.2 Preliminary test of catalyst-immobilized AuNPs 53.2.3 Properties of Mb-AuNP conjugates 55.2.4 Optimization of reagents in CL system 57.2.5 Effect of Mb-AuNP conjugates on CL intensity 60.2.6 Preliminary test of immunochromatographic strip for CymMV detection 61hapter 5 Conclusions 62eferences 6

Sensitive d-amino acid biosensor based on oxidase/peroxidase system mediated by pentacyanoferrate-bound polymer.

A sensitive d-amino acid oxidase (DAAO)/peroxidase (POD) bienzyme biosensor is constructed, in which pentacyanoferrate-bound poly(1-vinylimidazole) polymer (PVI[Fe(CN)5]) is selected as a mediator. Reductive current of PVI[Fe(CN)5] related to the H2O2 concentration generated in the DAAO reaction was measured at -0.1V vs. Ag|AgCl with DAAO/POD/PVI[Fe(CN)5]-modified electrode. The result revealed that PVI[Fe(CN)5] is suitable as a mediator for this bienzyme system due to its appropriate formal potential and its extremely low reactivity against DAAO. The stability of DAAO was improved by adding free flavin adenine dinucleotide and the electrode composition was optimized for the detection of d-alanine. Nafion and ascorbate oxidase-immobilized films worked successfully to prevent severe interference from uric acid and ascorbic acid. The low detection limits of d-alanine (2μM) and d-serine (2μM) imply its possibility for the determination of extremely low concentration of d-amino acids in physiological fluids. The proposed bienzyme biosensor is proved to be capable of detecting d-amino acids in urine

A Retrospective Review of the Prognostic Value of ALDH-1, Bmi-1 and Nanog Stem Cell Markers in Esophageal Squamous Cell Carcinoma

<div><p>Stem cell markers are upregulated in various cancers and have potential as prognostic indicators. The objective of this study was to determine the expression of three stem cell markers, aldehyde dehydrogenase 1 (ALDH-1), B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), and Nanog, in esophageal squamous cell carcinoma (ESCC) tissues. Immunohistochemistry was used to measure the expression of ALDH-1, Bmi-1, and Nanog in ESCC tissues from 41 patients who received pre-operative chemoradiation. We evaluated the relationship between expression of these markers, and clinicopathological features, tumor regression grade (TRG), and 5-year overall survival (OS). There were no significant associations of ALDH-1 or Bmi-1 expression with age, gender, clinical stage, and treatments (<i>p</i>>0.05). However, patients with Nanog-positive tumors were significantly older than those whose tumors were Nanog-negative (<i>p</i> = 0.033). TRG after treatment was significantly associated with expression of ALDH-1 (<i>p</i> = 0.001), Bmi-1 (<i>p</i> = 0.004), and Nanog (<i>p</i><0.001). Although OS was significantly better in patients with low TRGs (<i>p</i> = 0.001), there were no significant correlations between ALDH-1, Bmi-1, or Nanog with OS. Expression of ALDH-1, Bmi-1, and Nanog correlated with TRG, but not OS. Further large studies are necessary to fully elucidate the prognostic value of these stem cell markers for ESCC patients.</p></div