{"created":"2023-06-20T13:21:18.561018+00:00","id":1460,"links":{},"metadata":{"_buckets":{"deposit":"36d62555-a317-47e2-8518-af62b147a293"},"_deposit":{"created_by":21,"id":"1460","owners":[21],"pid":{"revision_id":0,"type":"depid","value":"1460"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001460","sets":["2:427:9"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"Md.,Abu,Sayed"}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"モハメド, アブ サイド"}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2009-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　　The study of protein-surface interactions represents one of the most important topics in the field of biomaterials. The immobilization of proteins on solid surfaces is an important step in biosensor fabrication as well as medical devices. Infrared spectroscopy is a powerful technique for the determination of conformation and orientation of lipids and proteins including membrane proteins, and of antibody-antigen reactions on solid surfaces. Infrared reflection absorption spectroscopy (IRRAS) is one of the FT-IR techniques for determination of biomaterials on IR reflective metals, and very few IRRAS systems are found to determine the adsorbate at solid-solution interfaces.<br />　　For my doctoral research work, I have constructed a new narrow gap infrared reflection absorption spectroscopy (NG-IRRAS) system with a prism/narrow solution layer/substrate arrangement, to which substrates for biosensors and biochips can be directly attached. Advantages of this new NG-IRRAS system over other conventional-IR systems are i) different IR reflective materials can be used as a substrate ii) having a sufficient large gap (〜8 μm ) to flow reagent solution, and iii) a solution injection system is included to introduce reagent solution onto the sample substrate from outside.  Another advantage of this IRRAS system is that this system can easily be rearranged to vacuum IRRAS system.  There were two problems to be solved for the standardization of this NG-IRRAS system, i) the baseline was fluctuated due to the change of solution layer thickness, and ii) sample biomaterials was adsorbed on prism surface.<br />　　In the first stage of my PhD work, I constructed the new NG-IRRAS system and investigated the conditions for the flat and stable baseline. Firstly, it was found that baseline was fluctuated due to the change of solution layer thickness. I developed a new sample holder for controlling the solution layer thickness between the prism and substrate surfaces (Fig. 1). (*Fig. 1 was abbreviated.)Thermal effects, adsorbed water on the entrance of optical components, bubbles in solution, and injection flow rate were considered as factors which distorted the baseline. I have investigated the experimental conditions and found that the following procedures are crucial for the stability of the baseline:　i) evacuation of the sample chamber at least for 6 h was necessary to minimize the adsorbed water vapor effect,ii) injection flow rate was kept less then 2 mL/h, iii) room temperature fluctuation was controlled within 1°C, and iv) Ni spacer thickness was 8 μm.<br />　　After the standardization of the NG-IRRAS, I started the IRRAS measurement of solid surfaces in three different conditions. i) The observation of CaF<small>2</small> prism surface in total internal reflection (TIR) mode to investigate the adsorbed biomaterials on prism surface. ii) The observation of biomaterials adsorbed at the interface between the sample substrate and solutions. Two IR-reflective substrates, Si wafer with buried metal layer (BML) and gold were used. iii) The observation of adsorbed biomaterials on the solid surface in vacuum. <br />　　Fibronectin (FN) and immunoglobulin G (IgG) were selected for observation at solid-solution interface. But, during the experiments, IR absorbance from the adsorbed biomaterials on the prism surface overlapped the IRRAS spectra of the biomaterials on the sample substrate. Thus, I controlled the adsorption of biomaterials on the prism surface by regulating the effects of salt and pH of the solution and by coating the CaF<small>2</small> prism with 2-methoxy-(polyethylene) oxypropyltrimethoxysilane (PEG). Interestingly, the adsorption tendency on the prism surface was completely opposite with salt effects between these two biomaterials (Fig. 2). (*Fig. 2(a),(b) were abbreviated.)<br />　　FN is easily adsorbed on SiO<small>2</small> surface and often used as an extra cellular matrix in the cell culture <br />on SiO<small>2</small> substrates. Therefore, FN was chosen in this work to detect at BML-solution interface. I have investigated the condition, in which FN adsorbed only on the BML surface, but not on the PEG-coated prism surface.  I found that FN adsorbed on the prism surface in D<small>2</small>O based phosphate buffered saline (PBS)solution, but ignorable adsorbed FN was found on the PEG-coated prism surface when pure D<small>2</small>O was used in the experiment (Fig. 2a).<br />　　The IRRAS of adsorbed FN was observed at BML-D<small>2</small>O interface using this condition. The protein amide I band appear in the range of 1600 - 1700 cm<sup>-1</sup> assigned to the C=O stretching. The fine structure in the amide I supplies the information of the protein secondary structure, because the peak shift of υ (C=O) due to the hydrogen bonding characteristics of the secondary structure, e.g. υ (C=O) of &beta;-sheet at  1613 -1638 cm<sup>-1</sup>, α-helix at 1645 - 1657 cm<sup>-1</sup> , and &beta;-turn at 1662 -1683 cm<sup>-1</sup>. The amide I band of FN was observed at approximately 1637 cm<sup>-1</sup> (&beta; sheet) with shoulders around 1671 and  1683 cm<sup>-1</sup> (β-turn)(Fig. 3a). (*Fig. 3(a).(b) were abbreviated.)　The ignorable conformational change of FN was found due to the adsorption on BML-D<small>2</small>O interface.<br />　　Immobilization of IgG on gold is an active research field for designing immunosensor for medical diagnostic purpose. The adsorption state of the IgG at the gold-solution interface was investigated by the NG-IRRAS. IgG was adsorbed on gold surfaces i.e. coated by the 16-mercaptohexadecanoic acid (MHA)-SAM and by the MHA-SAM activated by N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). It was also found that IgG was easily adsorbed on the PEG-coated prism surface in pure D<small>2</small>O, while IgG adsorption on the PEG-coated CaF<small>2</small> surface was suppressed sufficiently in NaCl/D<small>2</small>O (140 mM) and D<small>2</small>O based PBS solutions (Fig. 2b). I chose D<small>2</small>O based PBS solution containing 140 mM NaCl for IRRAS measurement of IgG at the gold-solution interface. <br />　　The IR spectra showed that IgG easily adsorbed on the MHA-SAM coated and activated-MHA-SAM coated gold surfaces. The conformation change of the IgG adsorbed on the MHA-SAM coated gold surface was ignorable when the IRRAS spectrum was compared with the FT-IR spectra of IgG dissolved in solution. Absorption spectra at the amide I band region of the IgG in the solution phase and at the solid-solution interface were measured with good reproducibility. The amide I band of the IgG molecule covalently bonded to the MHA-SAM (Fig. 3b) was quite similar to that of the IgG on the  MHA-SAM coated gold surface. The covalent bond is formed between the COOH-terminated MHA-SAM and the lysine residue of the IgG. Because of lysine residue distributes almost homogeneously on the IgG surface, the covalently bonded IgG had random orientation similar to the physisorbed IgG on MHA-SAM coated gold surface.<br />　　After the solid-solution interface IRRAS experiments, the same substrates were examined in the vacuum IRRAS. The amide I band shape was significantly different from that in the solution for both FN and IgG. This change of amide band is due to the denaturation of the proteins during the removal of water from the substrate surface.<br />　　As a whole, I have succeeded for the first time in constructing a new NG-IRRAS system having 8 μm gap to flow reagent solution for monitoring the chemical reaction. A specially designed sample holder is used to keep the gap constant during the injection of the reagent solution within a certain injection speed. Adsorption of proteins on the prism surface, which interferes with precise measurement, is suppressed using PEG-coating of the prism surface and controlling the solution pH and the effects of the salt. The amide I bands of the IgG molecules dissolved in the solution and covalently bonded to the COOH-terminated SAM surface at solid-solution interface have been clearly recorded for the first time. This new IRRAS instrument can be easily applicable in the haracterization of different antibody-antigen reactions on IR-reflective metal surfaces in physiological condition.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第1216号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"物理科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"07 構造分子科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2008"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"Md., Abu Sayed","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲1216_要旨.pdf","filesize":[{"value":"440.4 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/1460/files/甲1216_要旨.pdf"},"version_id":"cf3d1de4-7015-4b7d-8bd1-140dafd29b26"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲1216_本文.pdf","filesize":[{"value":"3.6 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/1460/files/甲1216_本文.pdf"},"version_id":"ac837e62-d80b-40c2-8e15-717f3a90013e"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Construction of New Infrared Reflection Absorption Spectroscopy (IRRAS) System for Solid-Solution Interface Biomaterials","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Construction of New Infrared Reflection Absorption Spectroscopy (IRRAS) System for Solid-Solution Interface Biomaterials"},{"subitem_title":"Construction of New Infrared Reflection Absorption Spectroscopy (IRRAS) System for Solid-Solution Interface Biomaterials　","subitem_title_language":"en"}]},"item_type_id":"1","owner":"21","path":["9"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-03-25"},"publish_date":"2010-03-25","publish_status":"0","recid":"1460","relation_version_is_last":true,"title":["Construction of New Infrared Reflection Absorption Spectroscopy (IRRAS) System for Solid-Solution Interface Biomaterials"],"weko_creator_id":"21","weko_shared_id":-1},"updated":"2023-06-20T16:03:45.742432+00:00"}