The investigation of molecularly-organized biocomposite nanostructures appears to drive a great deal of interest. In this regard, dendrimer-based biocomposite structures, organized mono- and multilayers, were developed for the catalytic and affinity biosensing.
For the catalytic biosensing inteface, a new approach to construct a multilayered enzyme film on the electrode surface was developed. The film was prepared by layer-by-layer depositions of poly(amidoamine) dendrimers and periodate-oxidized glucose oxidase. The cyclic voltammograms obtained from the Auelectrodes modified with Goudendrimer multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers, that is, to the amount of active enzyme immobilized. From the analysis of voltammetric signals, the coverage of active enzyme per layer during the muitilayer forming steps was estimated, which demonstrates that the multilayer is constructed in a spatially-ordered manner. Also with the eilipsomery, a linear increment of the film thickness was registered, supporting the formation of desired multilayer. As an extension of this research, poly(amidoamine) dendrimers having various degrees of modification with ferrocenyls were prepared and used for the biocomposite film. By alternate layer-by-layer depositions of ferrocenyl-tethered dendrimers with periodate-oxidized glucoseoxidase on Au, an electrochemically and enzymatically active multilayered assemblywas constructed. The resulting multilayered electrodes were electfochemically characterized, and the density of ferrocenyl groups, active enzyme coverage, and sensitivity were estimated. Dendrimers with 32% modifcatlon level was found to bean optimum from the analyses. With the prepared Fc(32%)-D, mono- and multilayered electrodes were constructed, and their olectrochernlcal and catalytic properties were characterized. From this result, it seems that the electrode sensitivityis directly controllable, and the multilayer-forming strategy with ferrocenyl-tethered dendrimers is useful for the construction of reagentless biosensors .
For the blospecific affinity-sensing interface, an affinity biosensor system based on avidin-biotin interaction was developed. As the building block of affinity sensing monolayer, G4 dendrimer having partial ferrocenyl-tethered suface groups was prepared and used. The surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was used for the affnity-sensing suface interacting with avidin .
Electrochemical signal from the affinity biosensor was generated by free enzyme in electrolyte, depending on the degree of coverage of the sensing surface with avidin and subsequent surface shielding.
The sensor signal decreased correlatively with increasing avidin concentration, and approached to a minimum when the sensing surface was fully covered. Detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5×$10^{-2}$ mol/㎠ on the avidin pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface. As an extension of this research, a new approach regarding the development of a repeatedly renewable affinity-sensing suface was presented. The surfaces were constructed with dendrimer monolayers, whose suface chain-end groups have been functionalized with biotin analogs having different affinity toward avidin, desthiobiotin .The functionaiized monoiayers provided an affinity recognition interface for avidin and further biospecific interactions with biotinylated molecules. The desthiobiotin-avidin assoclates undewent dissociation(displacement) reaction with free biotin treatment,and this renewed the affinity suface and provided the possibility of repeated utilization of the afinity-sensing suface. Biotinylated glucose oxidase, as a model compound for signal generation, was used for the association reaction onto the avidinpre-incubated surface, and voitammetric measurements were performed to track the reaction steps by registering the activity of associated enzyme. Efficient association/dissoclation reaction cycle traces were found, especially for the desthiobiotin amidocaproate modified electrodes, suggesting steric limitation regarding the ligand length for the biospecific interaction. Finally, the reversible affinity interactions at the functionalized electrode have been extended to antibodies. The surfaces were constructed with dendrimer monolayers, whose surFace chain-end groups have been double functionalized with biotins and ferrocenyls for the biospecific recognition and electron transfer. The functionalized monolayers provide a platform for biospecific recognition with monoclonal anti-biotin antibodies. The bound antibodies were dissociated with free biotin treatment, and the process renewed the affinity sufaces for repeated utilization. Tracking of the association/dissociation reaction cycles were peformed electrochemically, based on the shielding of electrode surface with bound antibody molecules and subsequent hindrance in electron transfer with free-diffusing signal generator and mediator Factors influencing the biospecific interactions and measurements were considered. With the results, continuous asseciation/dissociation reactions have been accomplished, holding great promise for the reversible affinity biosensing.
분자수준에서 규칙성을 갖는 생물조합형 나노구조의 형성 및 응용에 관한 관심이 날로 증대되고 있다. 이러한 관정에서 본 연구에서는 덴드라이머 고분자를 생물조합구조체의 구성블록으로 이용한 단일 막과 다중 막 구조의 형성과 이를 생체곡매 형과 생체특이결합형 바이오센서의 개발에 응용하고자 하는 연구를 수행하였다
첫째로, 생체촉매 형의 바이오센싱을 위하여, 효소 다중층을 전극표면에 구성하기 위한 방법론에 대한 연구가 수행되었다. 이를 통하여, 아민 반응기를 갖는 덴드라이머와 효소표면이 화학적으로 개질된 포도당 산화효소 간의 공유결합을 전극표면에서 유도하는 기술이 개발되었다. 전기화학적 측정과 ellipsometry를 통하여 덴드라이머에 의하여 매개된 효소 다중층의 형성이 확인되었고, 이 때 각 효소층은 활성이 극대로 유지된 상태로 효율적으로 형성됨을 검증하였다. 이 개념을 확장하여 덴드라이머를 다중층의 구성블록으로 이용함과 동시에 전자전달 매개체의 고정화담체로 이용하여 reagentless형의 바이오센서를 구성하기 위한 연구가 수행되었다. 이를 위하여 표면의 반응기가 일부 페로센으로 치환된 덴드라이머를 합성하여 사용하였다. 전자전달매개체의 치환정도에 따른 효소 다중층 전극의 특성을 전자전달매개체의 함량과 효소 다중층의 형성 그리고 전기적 활성 등의 관점에서 고찰하였고, 32%의 표면반응기가 치환된 덴드라이머를 이용한 효소다중층의 형성이 최적의 조건임을 결정할 수 있었다. 이를 통하여 제작된 Fc(32%)-D/GOx다중층을 이용하여 reagentless형의 생체촉매형 바이오센서를 효과적으로 개발할 수 있었다.
둘째로, 생체특이 결합형 바이오센서의 개발을 위하여 금 표면에서의 덴드라이머 단일막의 조성과 막 표면의 생체 리간드 수식에 관한 연구가 수행되었다. 우선 모델 시스템으로서 아비딘-바이오틴 결합을 고감도로 검출하기 위한 바이오센서의 개발연구가 수행되었다. 이를 통하여, 바이오틴으로 수식된 감응막의 아비딘과의 생체특이적 결합에 의해 유도되는 위치적 저해현상에 의한 반응검출을 실현하였다. 전기화학적 신호발생의 조건, 표지물질의 농도 등의 변수에 대한 최적화 실험을 통하여 약 4.5 pM의 측정한계에 이르는 고감도의 생체특이 결합형 바이오센서를 구현할 수 있었다. 또한 이 때 조성된 전극표면을 생체특이적 표지를 통하여 분석하여 분자수준으로 조밀한 단백질의 단일분자막이 효율적으로 구성됨을 검증하였다. 이 개념을 확장하여, 동일 전극표면에서 생체특이적 결합반응을 가역적으로 구현하기 위한 연구를 수행하였다. 이를 위하여 바이오틴에 의하여 환치 될 수 있는 유사분자인 desthiobiotin amidocaproate를 합성하고 덴드라이머 단일막에 기초한 아비딘 감응표면을 구성하였다. 결과적으로, desthtobiotin 리간드에 의한 아비딘의 생체특이적 결합과 바이오틴에 의한 감응막의 초기화 반응을 연속적으로수행할 수 있었으며, 따라서 재사용이 가능한 생체특이 결합형 바이오센서를 구현할 수있었다. 또한 이러한 시스템을 항원-항체반응에 적용하기 위한 연구를 수행하였고, 안티-바이오틴 항체를 이용한 실험을 통하여 가역적인 생체특이 결합반응이 가능한 바이오센서를 구현하였다.
