Control of Protein Binding at Interfaces: Gold Nanostructures and Affinity Peptide Labels

Controlling protein adsorption at solid surfaces is critical for a large variety of applications such as biocatalysis, biomedicine, food safety, and environmental monitoring. The importance of controlling the orientation and conformation of the protein at the surface has been recognized as key to the successful implementation of such applications. Metal nanostructure platforms have potential applications not only in protein-based biosensing but also in electronics and energy harvesting applications. This works investigates the specificity and selectivity of proteins binding to metal surfaces for incorporation into metal nanostructure arrays for such applications. The specificity and selectivity of the coupling in this case is achieved through a genetically engineered peptide tag with a high affinity for a gold surface. Controlling the protein orientation on selected areas of surfaces is challenging due to the inability to control the selectivity and specificity of the desired molecules. This work mainly focuses on the utilization of affinity peptides to control the protein orientation on selected areas of metal-organic hybrid films. Chapters 1 and 2 provide an introduction to the project and the methods employed. Investigations into the control of binding and orientations of a model protein (PutOX) at surfaces are described in Chapters 3 and 4. Chapter 3 describes the utilization of an affinity peptide towards the selective immobilization of proteins on surfaces. Affinity peptides are specific sequences of amino acids that have a high affinity for a material, and in this study, we investigate gold affinity peptides. The specific attachment of functionally active PutOx via gold affinity peptide sequence (AuBP) to gold surfaces has been demonstrated using QCM, activity assays, temperature treatment, and AFM investigations of coverage and shape of individual molecules. The temperature treatment studies show that the peptide tagged protein shows higher stability on the surface than in the solution. Chapter 4 reports the behavior of both wild type and PutOX-AuBP enzymes on a variety of surfaces including TSG (template stripped gold), mica, Si(111), OTS-Si-SAM, Graphite, COOH-TSG-SAM, and OH-TSG-SAM. This part of the study addresses the effect of properties of the surface in the attachment of protein in the presence of affinity peptide tags to understand the binding specificity and selectivity when using the AuBP. Once the selectivity and specificity of the gold binding peptide sequence towards the gold surface were demonstrated, methods to create Au nanostructures to bind the protein selectively on the metal nanostructures were required to create protein nanoarrays. Therefore, Chapters 5-7 investigate the fabrication of gold nanostructures. The development of gold plating solutions for electroless deposition is described in Chapter 5. The approach we used is electroless deposition (ELD), which is a well-established process on silicon and other semiconductor surfaces to deposit metal films. This chapter further discusses the effects of plating components and how self-assembled monolayers are used to selectively deposit metal on Si surfaces. Here, the effect of plating solution components, pH, and deposition time was studied to develop mild plating solutions for fabrication of gold nanostructures in Chapters 6 and 7. Two different methods for gold nanostructure fabrication are explored in these Chapters. Chapter 6 describes an AFM-based method for making gold nanowires using the combined techniques including self-assembled monolayer (SAM) resist formation, AFM nanoshaving, and electroless gold plating (whose deposition conditions were optimized in Chapter 5). Here, an OTS SAM on a silicon surface was utilized as a compelling molecular resist film for gold nanowire formation. The AFM nanoshaving was used to remove part of a molecular resist to expose the underlying silicon to facilitate gold nanostructure fabrication during ELD. In Chapter 7, a combination of particle lithography (PL), self-assembly, and electroless deposition was used to develop large, periodic arrays of gold nanodots on the silicon surface. The PL-based method addresses the limitations associated with the throughput of the AFM-based nanoshaving strategy discussed in Chapter 6. Here, the nanohole arrays are produced using a nanosphere mask and the formation of a self-assembled monolayer (SAM) film, and the nanohole array is then filled with metal via the ELD process. The metallic nanostructures developed using the PL provides the advantages of controllability of size and interparticle distance by changing the plating time and nanosphere diameter. The combination of developed nanostructures and the affinity peptide-tagged proteins can be used to develop materials for the fabrication of nanoscale bionanodevices, which display a range of surface chemistries in the device. The developed nanoarrays could be useful towards plasmonic biosensing applications, with localized plasmonic resonance wavelength tunability, as well as platforms capable of sensitive electrochemical detection. Finally, this dissertation addresses several critical concepts for biomaterials research including orientation control of proteins, precise placement of nanostructures and nanoarrays, modification of the surface, ease of fabrication, and cost. In the future, this study will be extended to develop multiple metal nanoarrays of silver and gold on the same surface to study cell signaling pathways or coupled enzyme reactions using multiple affinity peptides, which specifically bind to different metals (e.g., Au and Ag)

Fluorescent nanoparticles for sensing

Nanoparticle-based fluorescent sensors have emerged as a competitive alternative to small molecule sensors, due to their excellent fluorescence-based sensing capabilities. The tailorability of design, architecture, and photophysical properties has attracted the attention of many research groups, resulting in numerous reports related to novel nanosensors applied in sensing a vast variety of biological analytes. Although semiconducting quantum dots have been the best-known representative of fluorescent nanoparticles for a long time, the increasing popularity of new classes of organic nanoparticle-based sensors, such as carbon dots and polymeric nanoparticles, is due to their biocompatibility, ease of synthesis, and biofunctionalization capabilities. For instance, fluorescent gold and silver nanoclusters have emerged as a less cytotoxic replacement for semiconducting quantum dot sensors. This chapter provides an overview of recent developments in nanoparticle-based sensors for chemical and biological sensing and includes a discussion on unique properties of nanoparticles of different composition, along with their basic mechanism of fluorescence, route of synthesis, and their advantages and limitations

Mn3O4@CoMn2O4-CoxOy nanoparticles : partial cation exchange synthesis and electrocatalytic properties toward the oxygen reduction and evolution reactions

Mn3O4@CoMn2O4 nanoparticles (NPs) were produced at low temperature and ambient atmosphere using a one -pot two-step synthesis protocol involving the cation exchange of Mn by Co in preformed Mn3O4 NPs. Selecting the proper cobalt precursor, the nucleation of CoxOy crystallites at the Mn3O4@a CoMn2O4 surface could be simultaneously promoted to form Mn3O4@CoMn2O4-CoxOy NPs. Such heterostructured NPs were investigated for oxygen reduction and evolution reactions (ORR, OER) in alkaline solution. Mn3O4@ CoMn2O4-Cox0y NPs with [Co]/[Mn] = 1 showed low overpotentials of 0.31 Vat(-3) mA.cm(-2) and a small Tafel slope of 52 mV.dec(-1) for ORR, and overpotentials of 0.31 V at 10 mAPeer ReviewedPostprint (author's final draft

Nanopatterning with Photonic Nanojets: Review and Perspectives in Biomedical Research

: Nanostructured surfaces and devices offer astounding possibilities for biomedical research, including cellular and molecular biology, diagnostics, and therapeutics. However, the wide implementation of these systems is currently limited by the lack of cost-effective and easy-to-use nanopatterning tools. A promising solution is to use optical methods based on photonic nanojets, namely, needle-like beams featuring a nanometric width. In this review, we survey the physics, engineering strategies, and recent implementations of photonic nanojets for high-throughput generation of arbitrary nanopatterns, along with applications in optics, electronics, mechanics, and biosensing. An outlook of the potential impact of nanopatterning technologies based on photonic nanojets in several relevant biomedical areas is also provide

From Protein Building Blocks to Functional Materials

Proteins are the fundamental building blocks for high performance materials in nature. Such materials fulfil structural roles, as in the case of silk and collagen, and can generate active structures including the cytoskeleton. Attention is increasingly turning to this versatile class of molecules for the synthesis of next generation green functional materials for a range of applications. Protein nanofibrils are a fundamental supramolecular unit from which many macroscopic protein materials are formed. In this review, we focus on the multiscale assembly of such protein nanofibrils formed from naturally occurring proteins into new supramolecular architectures and discuss how they can form the basis of material systems ranging from bulk gels, films, fibers, micro/nanogels, condensates and active materials. We review current and emerging approaches to process and assemble these building blocks in a manner which is different to their natural evolutionarily selected role, but allows the generation of tailored functionality, with a focus on microfluidic approaches. We finally discuss opportunities and challenges for this class of materials, including applications that can be involved in this material system which consists of fully natural, biocompatible and biodegradable feedstocks yet has the potential to generate materials with performance and versatility rivalling that of the best synthetic polymers

이중블록공중합체와 마이셀로부터 합성한 정렬된 나노구조 배열의 대면적 그래핀 나노패터닝 응용

학위논문 (박사)-- 서울대학교 대학원 : 화학부 고분자화학전공, 2015. 8. 손병혁.그래핀이란 sp2 혼성 오비탈로 서로 결합된 탄소 원자들이 벌집 모양의 격자로 한 층에 배열된 평평한 물질을 말하며, 그래핀의 화학적, 물리학적 특성을 최적화하는 것은 신개념의 전자, 화학, 멤브레인 소자에 적용을 위해 매우 중요하다. 한편 그래핀을 나노미터 수준의 구조로 패터닝하게 되면 그래핀 기반의 소자들의 전하 이동 특성이 조절될 수 있음이 알려지게 되면서 그래핀에 나노제작 및 나노패터닝 기술을 적용하는 연구가 널리 진행되었다. 게다가 그래핀은 2차원 평면 물질의 구조적 특성을 가지므로 실리콘 기반의 반도체 공업에서 상용화된 가공 공법의 직접 적용이 가능하기 때문에 전자 빔 및 집속 이온 빔 리소그래피를 포함한 다양한 하향식 공정의 사용이 그래핀 패터닝에 접목될 수 있었다. 이러한 하향식 리소그래피 방법은 나노스케일의 패턴 사이즈를 정밀하게 조절할 수 있는 장점이 있기 때문에 나노패턴 그래핀의 다양한 우수한 물성을 발견함은 물론 해당 물성과 패턴 크기와의 관계까지도 정확하게 관찰하는 것이 가능하였다. 그러나 하향식 접근에서 활용되는 공정들은 고비용이고 많은 시간을 필요로 하는 복잡한 기술들이기 때문에 그래핀 패턴 형상의 유연성과 확장성 면에서 어려움이 많았다. 이에 반해 분자의 자기조립을 응용한 상향식 접근을 이용하면 대면적에서 나노구조 및 나노물질들을 효과적으로 제조할 수 있었기 때문에 저비용의 단순하고 쉬운 공정으로도 나노패턴 그래핀을 제조할 수 있다는 점에서 각광받기 시작했다. 여러 가지 상향식 방법들 중에서 이중블록공중합체를 통한 접근법은 다양한 물질들의 나노구조와 나노패턴을 제조할 수 있는 기술이라는 점에서 나노패턴 그래핀을 효과적으로 제조하는 데 응용될 수 있다. 이중블록공중합체는 서로 다른 두 고분자로 이루어져 있으며 자발적으로 조립하여 반복적인 나노구조를 형성하는데 나노구조의 크기와 모양은 공중합체의 분자량과 조성에 따라 조절할 수 있다. 또한 이중블록공중합체가 한쪽 블록만을 선택적으로 녹이는 용매에 용해되는 경우 녹는 블록으로 구성된 코로나와 안 녹는 블록으로 구성된 코어로 이루어진 구형의 마이셀을 얻을 수 있다. 이중블록공중합체와 마이셀은 고체 기판에 코팅되어 나노구조를 가지는 박막을 형성할 수 있는데 이는 식각 과정에 쓰이는 나노스케일의 리소그래피 마스크나 정렬된 무기 나노물질 배열을 제조하는 데 필요한 나노템플레이트로 쓰일 수 있다. 그래핀은 2차원의 원자 두께만큼이나 얇고 평평한 물질이기에 어떤 고체 기판 위에 있든지 이중블록공중합체와 마이셀의 박막을 코팅하기 용이하므로 그래핀이 없는 일반 기판에서 행하는 나노패터닝 공정들을 동일하게 진행할 수 있게 되는 것이다. 본 학위논문에서는 이중블록공중합체 접근법을 통한 대면적의 나노패턴 그래핀을 제조, 분석, 그리고 응용하는 것에 대해 다룬다. 먼저 1장에서 이 연구에 관한 배경 지식과 목표를 소개한 뒤, 이중블록공중합체와 마이셀 박막으로부터 정렬된 다양한 무기 물질의 배열을 제조하는 방법에 대해 2장에서 서술할 것이다. 제조된 나노구조는 나노템플리트, 에칭 마스크, 촉매 식각 구조로서 그래핀 나노패턴에 응용되는데, 공중합체의 분자량을 조절함으로써 제조되는 나노패턴의 크기와 간격을 나노수준에서 제어할 수 있다. 3장에서는 이들을 응용한 환원 산화 그래핀(rGO) 및 그래핀의 나노패터닝을 기술하며 나노입자로 장식된 rGO와 나노닷, 나노리본, 그리고 나노링 형태를 가진 rGO, 구멍 뚫린 그래핀의 배열 제조 실험 결과와 분석 내용을 포함하고 있다. 이중블록공중합체를 이용한 대면적의 그래핀 나노패터닝은 다양한 구조의 그래핀을 쉽고 효과적으로 제조할 수 있다는 장점이 있으므로 앞으로 전자, 화학, 생명, 환경 응용 소자 제작에 널리 활용될 것으로 기대된다.Controlling or optimizing the chemical and physical properties of graphene, a flat monolayer of sp2-bonded carbon atoms in a two-dimensional honeycomb lattice, has been of great importance for a variety of applications such as novel electronic, chemical, and membrane devices. Numerous results obtained by theoretical calculations and experimental observations have indicated that patterning graphene into nanometer-sized structures is useful for altering the physicochemical properties of graphene-based devices. Therefore, the use of modern nanofabrication and nanopatterning technologies with graphene materials has garnered much interest. Because the two-dimensional geometry of graphene is directly compatible with existing processing techniques used in the silicon-based semiconductor industry, a diversity of conventional top-down approaches including electron and focused ion beam lithography have been utilized for graphene nanopatterning. Top-down lithographic methods afford a precise means of controlling the feature sizes of nanoscaled patterns, resulting in the clear observation of various extraordinary properties of nanopatterned graphene as well as a deeper and more accurate understanding of the relationships between those properties and the pattern dimensions of graphene. However, the expensive, time-consuming, and sophisticated techniques associated with top-down approaches are major causes of the lack of flexibility and scalability of the graphene patterning process. In contrast, bottom-up approaches involving the use of molecular self-assembly processes, offer a wide range of opportunities to generate nanopatterned graphene through a low-cost, simple, and facile method, as these approaches effectively enable the fabrication of nanostructures and nanomaterials in large areas. The diblock copolymer approach, one of various bottom-up approaches, is a promising technique when used to generate nanostructures and nanopatterns of various materials. It can be used effectively to create nanopatterned graphenes. Diblock copolymers composed of two different polymers spontaneously assemble into periodic nanostructures, of which the size and morphology can be controlled by the molecular weight and composition of copolymers. In addition, when diblock copolymers are dissolved in a selective solvent for one of the blocks, spherical micelles with a soluble corona and an insoluble core can be obtained. The diblock copolymers and their micelles can be coated onto solid substrates to form nanostructured thin films, and they can be used as nanoscale lithographic masks for etching processes and nanotemplates to generate ordered arrays of inorganic nanomaterials. Because graphene is a two-dimensional, atomically flat material, a thin film of diblock copolymers and their micelles can be readily coated onto graphene which it is deposited onto any solid substrate, therefore guaranteeing further nanopatterning processes of the type used with substrates without graphene. This dissertation proposes the preparation, characterization and application of nanopatterned large-area graphene using the diblock copolymer approach. After introducing the background and objectives of this study in chapter I, the method used to fabricate diverse arrays of ordered inorganic nanostructures including nanoparticles (NPs), nanowires (NWs), and nanorings (NRs) from a thin film of diblock copolymers and their micelles is demonstrated in chapter II. Prepared nanostructures were used to prepare nanopatterned graphene as a nanotemplate, an etching mask, and a catalytic etcher, of which the feature sizes and spacings were effectively and accurately controlled at the nanoscale by adjusting the molecular weight of the copolymers. Chapter III presents the experimental results and the characterization of nanopatterned reduced graphene oxides (rGOs) and graphene, in this case rGOs decorated with nanoparticles, rGO nanodots, nanoribbons, and anti-NRs as well as nanoperforated graphene. This large-area nanopatterning technique using diblock copolymers provides a relatively facile and effective means of preparing various forms of nanostructured graphene in a controlled manner. Therefore, this methodology has immense potential when used with numerous electronic, chemical, biological, and environmental applications.Abstract i Table of Contents iv List of Figures vi List of Tables xxi List of Equations xxii Glossary xxiii Chapter I. Introduction 1. Graphene nanopatterning 1 2. Diblock copolymers and their micelles 6 3. Aims and objectives 10 II. Fabrication of arrayed inorganic nanostructures 1. Arrayed TiO2 nanostructures 11 2. Arrayed nanoparticles 21 3. Arrayed nanowires 34 4. Arrayed nanorings 48 5. Ordered complex arrays 69 III. Fabrication of nanopatterned graphene 1. Graphene decorated with arrayed nanoparticles 87 2. Graphene nanodot arrays 115 3. Graphene tailored by Pt nanostructures 131 IV. Summary and outlook 160 References 163 Appendices A. Electrochemical analysis 172 B. Photoluminescence from ZnO nanorods 175 C. Correlation analysis of nanoperforated graphene 177 Abstract in Korean 181 Curriculum Vitae 184Docto

Design, development and characterization of nanostructured electrochemical sensors

This is a publication-based thesis which focuses on the study of electrochemical microbiosensors for glucose detection. It investigates applications of a series of microfabricated gold electrodes based on several nanostructures in electrochemical biosensing technologies, embracing three major methodologies: direct electro-catalytic detection, enzymatic detection and dual-enzyme cascade detection. The study is described over five main chapters with a sixth providing a summary of the material presented and perspectives for the future. Chapter 1 provides an introduction to the field of the electrochemical biosensors with a specific focus on the chosen nanostructures and miniaturized systems, as well as a brief history of the biosensor. Chapter 2 presents results published in ACS Applied Nanomaterials, 2019, 2, 9, 5878-5889. It demonstrates the enzyme free detection of glucose via a direct electro-catalytic reaction. The miniaturized band array electrodes with specific width, length and inter-electrode-distance were integrated with homogeneously distributed copper foam nano dendrites. Such foam deposits presented for the first time at the micro scale were achieved using the in-situ hydrogen bubble template method. The resulting very high electroactive surface area of the porous foam deposits was one of the major advantages in terms of achieving superior performance from each micro band foam electrode towards glucose detection. Moreover, both sensors also showed a strong resistance to the poisoning effects of chloride ions and displayed excellent stability over a period of three months.Chapter 3 presents the first of t wo sets of results for the enzymatic detection of glucose, results published in Elsevier Electrochimica Acta, 2019, 293, 307-317. Chapter 4 then presents the second set of results on this topic which is published in and Elsevier Electrochimica Acta, 2019, 298, 97-105. The aim of these two chapters is to discuss the effect of miniaturization on the enzymatic biosensor performance which was studied in the presence of a carbon quantum dot (CQD) and gold nanoparticle nanohybrid system. CQDs, are a new class of carbon-based materials and have been used here for the first time as a matrix component integrated onto microfabricated gold electrode surfaces for enzyme immobilization and further miniaturization. The biosensors developed were studied by electrochemistry to investigate the analytical performance of each device. By scaling down the surface area of the biosensor, a 13-times increase in sensitivity was achieved towards glucose. Moreover both sensors-planar, micro disk array- exhibited excellent reproducibility, reusability and operational stability in terms of the performance of biosensors. Chapter 5 presents results published in RSC Analyst, 2020 (DOI: 10.1039/C9AN01664C). It demonstrates the operation of a dual-enzyme cascade which was constructed onto a micro band array electrode based on glucose oxidase and horseradish peroxidase enzymes. To achieve a very high surface area, a porous gold-foam was electrodeposited onto surface and then a second electrodeposition layer of chitosan and multi walled carbon nanotube nano-bio-composite. The micro band cascade scheme developed exhibited the highest sensitivity towards glucose detection in comparison to other systems reported in the literature. Chapter 6 provides an insight into the field of electrochemical biosensing with the support of the achievements presented in this thesis. Thus, by taking advantage of the available system, this chapter discusses the possible future applications of the electrochemical biosensors. The thesis then ends with section 7 which presents some Appendices

Fabrication of Horizontal Silicon Nanowires Using a Thin Aluminum Film as a Catalyst

Silicon nanowires have been the topic of research in recent years for their significant attention from the electronics industry to grow even smaller electronic devices. The semiconductor industry is built on silicon. Silicon nanowires can be the building blocks for future nanoelectronic devices. Various techniques have also been reported in fabricating the silicon nanowires. But most of the techniques reported, grow vertical silicon nanowires. In the semiconductor industry, integrated circuits are designed and fabricated in a horizontal architecture i.e. the device layout is flat compared to the substrate. When vertical silicon nanowires are introduced in the semiconductor industry, a whole new architecture is needed to fabricate an electronic device. If the silicon nanowires can be grown horizontally, it will be much easier to incorporate these nanowires in the current architecture. In this thesis, horizontal silicon nanowires were grown on top of a silicon substrate in a bottom up approach. A thin layer of pure aluminum was used as a catalyst to grow the silicon nanowires. In this process, silicon from the substrate itself acts as a source to grow the nanowires. A device cannot be fabricated if the silicon nanowires are in full contact with the underlying silicon substrate. Therefore, in the later part, these silicon nanowires were grown on top of an oxide layer using the same conditions. Several windows were etched in the oxide layer with variable oxide widths to observe the growth of these nanowires

Plasma Nanoscience: from Nano-Solids in Plasmas to Nano-Plasmas in Solids

The unique plasma-specific features and physical phenomena in the organization of nanoscale solid-state systems in a broad range of elemental composition, structure, and dimensionality are critically reviewed. These effects lead to the possibility to localize and control energy and matter at nanoscales and to produce self-organized nano-solids with highly unusual and superior properties. A unifying conceptual framework based on the control of production, transport, and self-organization of precursor species is introduced and a variety of plasma-specific non-equilibrium and kinetics-driven phenomena across the many temporal and spatial scales is explained. When the plasma is localized to micrometer and nanometer dimensions, new emergent phenomena arise. The examples range from semiconducting quantum dots and nanowires, chirality control of single-walled carbon nanotubes, ultra-fine manipulation of graphenes, nano-diamond, and organic matter, to nano-plasma effects and nano-plasmas of different states of matter.Comment: This is an essential interdisciplinary reference which can be used by both advanced and early career researchers as well as in undergraduate teaching and postgraduate research trainin