Development of Screen-Printed Biosensors Based on Chitosan-Multiwalled Carbon Nanotube-Platinum Nanocomposite

Since the discovery of carbon nanotubes, they has attracted the attention of many scientists worldwide. The small dimensions, remarkable physical properties, and the enhancement of electron reactions made them a very unique material with a whole range of promising applications. Chitosan is a transformed polysaccharide obtained by deacetylation of natural chitin. Chitosan has attracted increasing attention as a biomaterial for immobilizing enzyme through the formation of polyelectrolyte complexes between the enzymes and polysaccharide chains of the chitosan. Platinum (Pt) nanoparticles can catalyze some specific substances and decrease the working potential. The aim of this study is to investigate biosensors based on chitosan-multiwalled carbon nanotubes- enzyme nanobiocomposite, which was modified with Pt nanoparticles prepared by electrodeposition on chitosan-multiwalled carbon nanotubes nanocomposite.The properties of chitosan-Pt nanoparticles-multiwalled carbon nanotubes-enzyme nanobiocomposite were characterized by atomic force microscopy(AFM), scanning electron microscopy (SEM), X-ray diffraction (XRD), cyclic voltammetry (CV), and ampermetry. The incorporation of enzymes within chitosan-Pt nanoparticle-multiwalled carbon nanotubes composite is investigated by UV/VIS. The chitosan-Pt nanoparticles-multiwalled carbon nanotubes-enzyme nanobiocomposite can be applied to biosensor after immobilizing biomaterials such as glucose oxidase, alcohol dehydrogenase, cholesterol oxidase, lactate dehydrogenase, ssDNA, and tyrosinase. Finally, we hope to use the resulting chitosan-Pt nanoparticle-multiwalled carbon nanotubes-enzyme nanobiocomposite to fabricate disposable electrodes by a screen-printing technique.本研究計畫主要希望將奈米鉑粒子以電化學沈積法修飾在幾丁聚醣(chitosan)-多層壁奈米碳管(multiwalled carbon nanotubes)複合材料上，並將此幾丁聚醣-多層壁奈米碳管-奈米鉑粒子複合材料應用於生物感測器(biosensor)上，以提高其生物感測器靈敏度。採用奈米碳管是希望藉由奈米碳管的電催化特性，提高受測物質的氧化/還原性質，而幾丁聚醣則為一無毒性與生物相容性高分子，已廣泛應用於生醫材料，加入奈米鉑粒子在於能催化某些特定反應和降低工作電壓以避免干擾物。幾丁聚醣-多層壁奈米碳管-奈米鉑粒子複合材料的特性，將以原子力顯微鏡(atomic force microscopy)與電子顯微鏡(scanning electron microscopy)觀察奈米鉑粒子於多層壁奈米碳管的分佈情形，使用循環伏安法(cyclic voltammetry)探討幾丁聚醣-多層壁奈米碳管-奈米鉑粒子複合材料的電催化性質，並和無奈米鉑粒子的幾丁聚醣-多層壁奈米碳管複合材料比較反應物質在溶液中質量傳遞與電子傳遞行為。以紫外光/可見光(UV/VIS)探討各種酵素是否成功包埋在幾丁聚醣-多層壁奈米碳管-奈米鉑粒子複合材料中，並以安培法(ampermetry)探討幾丁聚醣-多層壁奈米碳管-奈米鉑粒子複合材料的感測靈敏度與偵測極限，其製備完成的幾丁聚醣-多層壁奈米碳管-奈米鉑粒子-酵素生物感測器將應用於偵測葡萄糖(glucose)、膽固醇(cholesterol)、乳酸(lactate)、乙醇(ethanol)、DNA等生物分子。最後期望藉由高分子、多層壁奈米碳管、奈米鉑粒子及酵素的結合，利用網版印刷技術(screen-printing technique)發展出靈敏度高、穩定性好及可拋棄式的生物感測器

97年度育成中心知識服務環境建構計畫動態整合服務功能之育成支援中心

國立中興大學創新育成中心為本育成支援中心之主要執行者，以整合資源與知識管理為基礎，而為提升育成產業之服務品質，加強其服務能力，本計畫朝加速促成知識與資訊平臺融合方向邁進，藉由資訊科技整合育成相關知識，以建構育成產業的知識服務環境，並結合區域內各育成中心之特有資源及共通性專業服務資源，加強創新創業的專業輔導能量與品質。因此，將全程計畫分為三階段執行：第一階段以建構基礎技術交流與服務網絡支援平臺、整合育成中心內部專長暨相關資源、服務區域內育成中心及育成企業；第二階段建置資訊平臺、整合育成中心外部相關資源、蓄

The Study of Multiwalled Carbon Nanotube Deposed with Conducting Polymer for Sensor

The aim of this study is to investigate the preparation of multiwalled carbon nanotube deposed with conducting polymer for sensors. Advantages of the electrochemical formation of conducting polymers films are (i) an easy preparation procedure, (ii) accurate control of the polymer thickness via the electrical charge passed during the film formation process, (iii) localization of the electrochemical reaction exclusively on the electrode surface allowing precise modification of electrodes and surfaces of complex geometry, and (iv) the possibility to build up multi-layer structures. These have made the conducting polymers to be utilized in the development of sensors.Since the discovery of carbon nanotubes, there has been attracted the attention of many scientists worldwide. Carbon nanotubes can be divided into two categories. One is the multiwall carbon nanotubes and the other is the single-wall carbon nanotubes. The small dimensions, remarkable physical properties and the enhancement of electron reactions made them a very unique material with a whole range of promising applications. They can be applied on biological cell electrode, catalysts, nanoscale electronic and mechanical systems, scanned probe microscope and electron field emission tips.The potential applications of carbon nanotubes in chemical sensors would greatly benefit from the ability of carbon nanotubes to promote the electron-transfer reactions of molecules. The difficulty for developing a carbon nanotubes-based sensor is the insolubility of carbon nanotubes in all solvents. The challenge of solubilizing carbon nanotubes can be resolved by covalent modification or noncovalent functionalization. In particular, the entrapment of carbon nanotubes in polymer matrix has shown useful for improving their solubility.The sensors made by this study would be able to detect heavy metals in the presence of surfactants and biomolecules, for instance, glucose.本計畫為發展以導電高分子被覆蓋在奈米碳管作為感測器之研究，以導電高分子作為感測器具有下列幾項特點：(1).可以將導電高分子以任何形狀和大小經由電化學聚合法被覆在電極上。(2).電化學聚合法過程中經由控制流過電極電量進而決定高分子膜厚度。(3).可以將導電高分子依需要作必要改質(modification)。導電高分子在摻雜(doped)和去摻雜(undoped)之間會造成其導電度和其他性質改變，利用此一性質可以使用在生物感測器上。採用奈米碳管是希望藉由奈米碳管的電催化活性，提高受測物質的氧化/還原性質，產生高的電流密度，並提供穩定的電流訊號。將導電高分子被覆在奈米碳管是希望藉由導電高分子的被覆，能減少溶液中雜質如界面活性劑等吸附在電極表面，造成電流訊號降低，提高偵測溶液中重金屬含量時的靈敏度。另外，利用導電高分子和酵素在中性水溶液中的相容，而直接以電化學聚合法聚合在奈米碳管電極上，在聚合過程中將酵素包圍在導電高分子本體內作為生物感測器，期望藉由導電高分子、奈米碳管及酵素的結合，能成功的發展出靈敏度高、穩定性好的生物感測器

Fabrication of Nanosensor in Nanochannels Produced by AFM Lithography

本計畫為研究以掃瞄式探針顯微術製備奈米圖案的模板，並以此模板進行電化學聚合反應製備導電高分子奈米結構，並以此導電高分子奈米結構做為奈米感測器探討。本計畫不採用商業化之奈米孔洞薄膜，而使用掃瞄式探針顯微術製備奈米圖案的模板，並使用電化學聚合則是因為其具備下列優點：(1).相較於傳統微影術，掃瞄式探針顯微術可非常容易且快速的製作出奈米尺度的模板，且與商業化的薄膜比較，是相當便宜的。(2).模板的形狀可以是點、線、陣列，甚至其它形狀，可依實驗需求而製作。(3).利用電化學聚合的導電高分子奈米結構與化學法聚合比較時，可以快速的製作且形狀、厚度、均勻性皆可容易的控制。(4).只要替換其它電化學聚合物質，就可以製作出其他物質的奈米結構，具有相當大的潛力可以製備出金屬或半導體的奈米材料。導電高分子在摻雜(doped)和去摻雜(undoped)之間會造成其導電度和其他性質改變，利用此一性質可以使用在生物感測器上。另外，利用導電高分子和酵素在中性水溶液中的相容，而直接以電化學聚合法聚合於奈米尺度的圖案上，在聚合過程中將酵素包圍在導電高分子本體內作為奈米生物感測器，期望藉由成功製備的導電高分子和其他物質的奈米結構，能研究發展出微小化的奈米感測器，用來偵測如重金屬、葡萄糖和膽固醇等

全氟磺酸聚合物-石墨烯奈米生物高分子薄膜開發與在生物感測器之應用

本研究計畫主要係探製備討全氟磺酸聚合物(Nafion)-石墨烯(Graphene)奈米生物複合薄膜並修飾於玻璃碳電極(glassy carbon electroe, GCE)上，製備成化學感測器和生物感測器。希望藉由Nafion-Graphene奈米複合薄膜之電催化與增加電子傳遞速度的特性來增加感測器對受測物的各種電化學表現。本研究計畫為三年計畫，第一年計畫以Nafion-Graphene奈米複合薄膜修飾於GCE上，接著用循環伏安法(cyclic voltammetry)及方波伏安法(square wave voltammetry)分別對阿斯匹靈與乙醯胺酚進行偵測以得到最佳靈敏度與偵測極限，再以此修飾電極在最佳參數下分別偵測市面上販售含阿斯匹靈與乙醯胺酚的藥品來確保其準確度。在第二年將以辣根過氧化酵素(horseradish oxidoreductase, HRP)固定於Nafion-Graphene奈米複合薄膜並修飾於GCE上，製備出具有直接電子傳遞特性的過氧化氫(H2O2)感測器，再利用此電極以循環伏安法及安培法對過氧化氫進行偵測以得到此感測器效能之各參數最適值。最後一年將把DNA探針固定於Nafion-Graphene奈米複合薄膜並修飾於GCE上，用微分脈衝伏安法(differential pulse voltammetry)探討與標的DNA雜交時所偵測到的電流及電位訊號，以得到各種最佳之效能參數並進行機制探討。而各階段Nafion-Graphene結合酵素或DNA探針的表面性質將以原子力顯微鏡(atomic force microscopy, AFM)及掃描式電子顯微鏡(scanning electron microscope, SEM)對其表面進行觀察，期望藉由本計畫的執行能將Nafion-Graphene奈米生物複合薄膜廣泛利用於電化學生物感測器的應用。Graphene has been attracted great attention in many fields such as supercapacitors, hrdrogen storage and chemical sensors due to its high surface area, remarkable electrical conductivity and high thermal conductivity. Graphene has the ability to promote electron transfer when used as the electrode material, which provides a new way of designing novel electrochemical sensors, biosensors and DNA sensors. In this proposal, Nafion was used as a dispersant to disperse graphene in aqueous solution. In addition, Nafion can also increase the immobilization stability of graphene with enzyme on GCE surface due to its excellent film forming ability. And the feasibility to use Nafion-graphene nanocomposite for determination of acetylsalicylic acid, acetaminophen, hydrogen peroxide, and DNA is proposed. The surface morphology of the resulting film will be investigated by scanning electron microscopy and atomic force microscopy. The incorporation of horseradish peroxidase within Nafion-graphene nanocomposite will be investigated by electrochemical impedance spectroscopy. The electroanalytical response of the Nafion-graphene nanocomposite film modified electrode will be evaluated by cyclic voltammetry, square wave voltammetry, differential pulse voltammetry and amperometry to find out the optimal values

Nanoelectrode Arrays for Biosensing

奈米電極(nanoelectrode)具有少量樣品需求、質傳速度快、和低界面電容等優點，適合應用於電化學領域。因此，利用微電極的應用概念，我們將以奈米電極陣列發展生物感測器，以求更高的靈敏度與應用性。本計畫為三年計畫，第一年運用西班牙國家高等科學研究委員會合作團隊所製作出的奈米電極陣列，進行電極特性探討。首先，將以原子力顯微鏡(atomic force microscopy)與掃描式電子顯微鏡(scanning electron microscopy)觀察奈米電極陣列表面形貌。接著再使用電化學方法，探討對此奈米電極陣列對過氧化氫和β-菸鹼醯胺腺嘌呤二核甘酸的電行為，並評估此奈米電極陣列應用於生物感測器的可行性。第二年則是利用將奈米電極陣列製作葡萄糖感測器和酒精感測器。以導電高分子將酵素分散，再以電化學聚合方式將葡萄糖氧化酵素與高分子一起固定修飾於奈米電極表面，並以傅立葉紅外線光譜儀(FT-IR)確認酵素固定。接著使用AFM和SEM觀察生物複合材料的表面性質，另外再以循環伏安法(cyclic voltammetry)和安培法(amperometry)探討高分子-酵素生物複合材料的電化學催化性質，並對葡萄糖生物感測器和酒精感測器之電流訊號做探討，找出影響葡萄糖感測器效能的各參數並將之最適化。本計畫第三年將藉著先前兩年的技術與經驗，將生物感測器更延伸應用於DNA生物感測器。找出能有效地分散DNA的導電高分子，並以電化學聚合方式將DNA和高分子一起修飾奈米電極表面，再以紫外光/可見光(UV-Vis)確認DNA成功固定。此種生物複合材料表面以AFM觀察DNA分散狀態及其膜的均勻度。以循環伏安法和安培法探討其電化學催化性質，並對DNA生物感測器之電流訊號做探討，找出影響DNA感測器的各個參數並使之最適化。There are many advantages about the nanoelectrode electrode when employed in electrochemical application such as small sample volume, fast mass transport rate, low interfacial capacitance, and so on. In principle by decreasing electrode size, study of faster electrochemical reaction should be possible. Due to the electron transfer process is less likely to be limited by the mass transport of reactant to the electrode surface at fast mass transport rate. Based on those benefits, we will develop the biosensor by using the nanoelectrode arrays which is provided by Instituto de Microelectronica de Barcelona, CNM-CSIC. At first, to estimate the probability for application of nanoelectrode arrays, we investigate the morphology of the nanoelectrode arrays by atomic force microspcopy (AFM) and scanning electron microscope (SEM). The electrocatalytic behavior of the nanoelectrode arrays toward electrochemical oxidation of hydrogen peroxide and β-nicotinamide adenine dinucleotide (NADH) is studied. According to the above testing, we fabricate biosensor for detecting glucose and alcohol by nanoelectrode arrays. The electroananlytical response of the modified nanoelectrode arrays is investigated by cyclic voltammetry (CV) and amperometry method to find out the optimal value. To further application, we will fabricate DNA biosensor which is important to the diagnosis and treatment of genetic diseases. The influence of experimental parameters are explored to optimize the electroanalytical performance of the DNA sensor. The DNA and enzyme are incorporated into conducting polymers by entrapment of DNA probes and enzyme within electropolymerized polymer film. The aim of this plan is to achieve the biosensors with faster response time, simple preparation, and highly sensitivity

Development and Biosensor Applications of Chitosan-Multiwalled Carbon Nanotubes Nanobiocomposite

本研究計畫主要希望能開發將多層壁奈米碳管懸浮於含有幾丁聚醣的水溶液中,而得到一均勻分散多層壁奈米碳管-幾丁聚醣溶液, ,採用奈米碳管是希望藉由奈米碳管的電催化特性,提高受測物質的氧化/還原性質,而幾丁聚醣則為一無毒性與生物相容性高分子，已廣泛應用於生醫材料。將多層壁奈米碳管-幾丁聚醣均勻溶液修飾在玻璃碳電極上(glassy carbon electrode),製備多層壁奈米碳管-幾丁聚醣奈米高分子複合材料，以原子力顯微鏡觀察多層壁奈米碳管-幾丁聚醣奈米高分子複合材料成膜後均勻性，使用循環伏安法(cyclic voltammetry)探討多層壁奈米碳管-幾丁聚醣奈米高分子複合材料的電催化性質,利用交流阻抗儀(impedance)探討離子在電化學反應過程中在多層壁奈米碳管-幾丁聚醣奈米高分子複合材料中傳遞機構,將多層壁奈米碳管-幾丁聚醣奈米高分子複合材料和傳統電極比較反應物質在溶液中質量傳遞和電子傳遞行為。本研究將進一步在這均勻分散的多層壁奈米碳管-幾丁聚醣溶液中加入各種酵素製作成一新穎的奈米生物高分子薄膜,做為生物感測器的研究,以紫外光/可見光(UV/VIS)探討各種酵素成功包埋在多層壁奈米碳管-幾丁聚醣奈米高分子複合材料中，以安培法(amperometry)探討各種生物感測之靈敏度和偵測極限,以石英震盪微量天平(quartz crystal microbalance, QCM)探討酵素和待測物質反應時重量變化進而探討其反應機制。在多層壁奈米碳管-幾丁聚醣-酵素奈米生物高分子複合材料中引入金屬奈米顆粒以提高其靈敏度，將製備完成的各種生物感測器應用於偵測溶液中的葡萄糖(glucose)、膽固醇(cholesterol)、乳酸(lactate)、酚(Phenol)、乙醇(ethanol)、DNA等生物分子的含量。期望藉由高分子、多層壁奈米碳管及酵素的結合,能成功發展出靈敏度高、穩定性好的生物感測器

Biosensors Based on Multiwalled Carbon Nanotubes-Poly(Vinyl Alcohol) Nanoccmposite

本研究計畫主要希望能發展將多層壁奈米碳管懸浮於含有聚乙烯醇的水溶液中,而得到一均勻分散溶液,再將多層壁奈米碳管-聚乙烯醇均勻溶液修飾在玻璃碳電極上(glassy carbon electrode),利用循環伏安法(cyclic voltammetry)探討其電催化性質,利用交流阻抗法(impedance)探討離子在電化學反應過程中傳遞機構,並和傳統電極比較反應物質在溶液中質傳和電子傳遞行為,採用奈米碳管是希望藉由奈米碳管的電催化特性,提高受測物質的氧化/還原性質,而聚乙烯醇則為一無毒性與生物相容性水膠。再進一步利用這均勻分散的多層壁奈米碳管-聚乙烯醇溶液中加入各種酵素製作成一新穎的奈米生物薄膜,進而修飾在電極上, 做為生物感測器的研究,以安培法(amperometry)探討各種生物感測之靈敏度和偵測極限,以石英震盪微量天平(quartz crystal microbalance, QCM)探討酵素和待測物質反應時重量變化進而探討其反應機制。將製備完成的各種生物感測器偵測溶液中的葡萄糖(glucose)、膽固醇(cholesterol)、乳酸(lactate)、酚(Phenol)、乙醇(ethanol)等生物分子的含量。期望藉由水溶性高分子、多層壁奈米碳管及酵素的結合,能成功發展出靈敏度高、穩定性好的生物感測器