Programmable interactions with biomimetic DNA linkers at fluid membranes and interfaces

At the heart of the structured architecture and complex dynamics of biological systems are specific and timely interactions operated by biomolecules. In many instances, biomolecular agents are spatially confined to flexible lipid membranes where, among other functions, they control cell adhesion, motility and tissue formation. Besides being central to several biological processes, \emph{multivalent interactions} mediated by reactive linkers confined to deformable substrates underpin the design of synthetic-biological platforms and advanced biomimetic materials. Here we review recent advances on the experimental study and theoretical modelling of a heterogeneous class of biomimetic systems in which synthetic linkers mediate multivalent interactions between fluid and deformable colloidal units, including lipid vesicles and emulsion droplets. Linkers are often prepared from synthetic DNA nanostructures, enabling full programmability of the thermodynamic and kinetic properties of their mutual interactions. The coupling of the statistical effects of multivalent interactions with substrate fluidity and deformability gives rise to a rich emerging phenomenology that, in the context of self-assembled soft materials, has been shown to produce exotic phase behaviour, stimuli-responsiveness, and kinetic programmability of the self-assembly process. Applications to (synthetic) biology will also be reviewed.Comment: 63 pages, revie

The effect of tethers on artificial cell membranes: A coarse-grained molecular dynamics study

© 2016 Hoiles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Tethered bilayer lipid membranes (tBLMs) provide a stable platform for modeling the dynamics and order of biological membranes where the tethers mimic the cytoskeletal supports present in biological cell membranes. In this paper coarse-grained molecular dynamics (CGMD) is applied to study the effects of tethers on lipid membrane properties. Using results from the CGMD model and the overdamped Fokker-Planck equation, we show that the diffusion tensor and particle density of water in the tBLM is spatially dependent. Further, it is shown that the membrane thickness, lipid diffusion, defect density, free energy of lipid flip-flop, and membrane dielectric permittivity are all dependent on the tether density. The numerically computed results from the CGMD model are in agreement with the experimentally measured results from tBLMs containing different tether densities and lipids derived from Archaebacteria. Additionally, using experimental measurements from Escherichia coli bacteria and Saccharomyces Cerevisiae yeast tethered membranes, we illustrate how previous molecular dynamics results can be combined with the proposed model to estimate the dielectric permittivity and defect density of these membranes as a function of tether density

Direct measurement of DNA-mediated adhesion between lipid bilayers

Multivalent interactions between deformable mesoscopic units are ubiquitous in biology, where membrane macromolecules mediate the interactions between neighbouring living cells and between cells and solid substrates. Lately, analogous artificial materials have been synthesised by functionalising the outer surface of compliant Brownian units, for example emulsion droplets and lipid vesicles, with selective linkers, in particular short DNA sequences. This development extended the range of applicability of DNA as a selective glue, originally applied to solid nano and colloidal particles. On very deformable lipid vesicles, the coupling between statistical effects of multivalent interactions and mechanical deformation of the membranes gives rise to complex emergent behaviours, as we recently contributed to demonstrate [Parolini et al., Nature Communications, 2015, 6, 5948]. Several aspects of the complex phenomenology observed in these systems still lack a quantitative experimental characterisation and fundamental understanding. Here we focus on the DNA-mediated multivalent interactions of a single liposome adhering to a flat supported bilayer. This simplified geometry enables the estimate of the membrane tension induced by the DNA-mediated adhesive forces acting on the liposome. Our experimental investigation is completed by morphological measurements and the characterisation of the DNA-melting transition, probed by in-situ F\"{o}rster Resonant Energy Transfer spectroscopy. Experimental results are compared with the predictions of an analytical theory that couples the deformation of the vesicle to a full description of the statistical mechanics of mobile linkers. With at most one fitting parameter, our theory is capable of semi-quantitatively matching experimental data, confirming the quality of the underlying assumptions.Comment: 16 pages, 7 figure

DNA-Mediated Self-Assembly of Artificial Vesicles

Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time.In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts.This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and targeting of long-lived, pharmacologically inert prodrugs and their conversion to short-lived, active drug molecules directly at the site of action may be accomplished if multi-vesicle assemblies of predefined architecture are used

Supported Lipid Membranes and Their Use for the Characterization of Biological Nanoparticles

Biological nanoparticles (BNPs) are nano-sized lipid vesicles of biological origin, which are involved in multiple biological processes. BNP characterization techniques are critical for improving the understanding of how these particles contribute to cellular communication, viral infections and drug-delivery applications. However, due to their small size (between 50 and 200 nm in diameter) and molecular heterogeneity, quantitative characterization of their physical, chemical and biological properties is demanding, especially since their large structural and compositional heterogeneity calls for methods with single nanoparticle resolution. To address this challenge, work in this thesis has been focused on investigating and using supported lipid bilayers (SLBs) and their two-dimensional fluidity as a platform for nanoparticle characterization. To investigate SLBs, we combined confocal microscopy with microfluidics to identify the mechanisms by which lipid vesicles are spontaneously converted into various types of planar membranes on a multitude of surfaces (Paper\ua0I) and found that most of the studied materials can support lipid film formation. In the context of SLB formation, specific focus was put on using total internal reflection fluorescence (TIRF) microscopy to monitor the kinetics of vesicle adsorption, rupture and spreading of individual SLB patches on glass (Paper\ua0II), revealing that the SLB formation process was driven by the autocatalytic growth and merger of multiple small SLB patches at appreciably high vesicle coverage. TIRF was also successfully employed to monitor lipid-enveloped drug permeation through an SLB formed on a mesoporous silica thin film (Paper III). The insights gained from investigating SLBs was also used for in depth characterization of BNPs using the surface-based flow-nanometry method, allowing for independent determination of size and fluorescence emission of individual BNPs tethered to a laterally fluid SLB formed on the floor of a microfluidic channel. This way we could demonstrate that the fluorescence emission from lipophilic dyes depends in a non-trivial way on nanoparticle size, and varies significantly between the different types of BNPs (Paper IV). The flow-nanometry concept was also used to elucidate the effect of vesicle size on their diffusivity on the SLB in the limit of few tethers (Paper V). The insights gained in this thesis work on lipid self-assembly at different surfaces and the possibility to use SLBs on silica for in-depth characterization of BNPs demonstrate this as a promising approach in the field of single nanoparticle analytics, which in future work will be possible to extend into a novel means to probe interactions between BNPs and cell-membrane mimics representing a near-native situation

Recent Advances in Hybrid Biomimetic Polymer-Based Films: from Assembly to Applications

Biological membranes, in addition to being a cell boundary, can host a variety of proteins that are involved in different biological functions, including selective nutrient transport, signal transduction, inter- and intra-cellular communication, and cell-cell recognition. Due to their extreme complexity, there has been an increasing interest in developing model membrane systems of controlled properties based on combinations of polymers and different biomacromolecules, i.e., polymer-based hybrid films. In this review, we have highlighted recent advances in the development and applications of hybrid biomimetic planar systems based on different polymeric species. We have focused in particular on hybrid films based on (i) polyelectrolytes, (ii) polymer brushes, as well as (iii) tethers and cushions formed from synthetic polymers, and (iv) block copolymers and their combinations with biomacromolecules, such as lipids, proteins, enzymes, biopolymers, and chosen nanoparticles. In this respect, multiple approaches to the synthesis, characterization, and processing of such hybrid films have been presented. The review has further exemplified their bioengineering, biomedical, and environmental applications, in dependence on the composition and properties of the respective hybrids. We believed that this comprehensive review would be of interest to both the specialists in the field of biomimicry as well as persons entering the field

Tunable cell-surface mimetics as engineered cell substrates

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed

Plasmonic atoms and molecules for imaging and sensing

Nanoscale structures play a fundamental role in diverse scientific areas, including biology and information technology. It is necessary to develop methods that can observe nanoscale structures and dynamic processes that involve them. Colloidal plasmonic nanoparticles (plasmonic “atoms”) and their clusters (plasmonic “molecules”) are nanoscale objects with remarkable optical properties that provide new opportunities for sensing and imaging on the relevant length and time scales. Many biology questions require optically monitoring of the dynamic behavior of biological systems on single molecule level. In contrast to the commonly used fluorescent probes which have the problem of bleaching, blinking and relatively weak signals, plasmonic probes display superb brightness, persistency and photostability, thus enable long observation time and high temporal and spacial resolutions. When plasmonic atoms are clustered together, their resonances redshift while the intensities increase as a result of plasmon coupling. These optical responses are dependent on the interparticle gaps and the overall geometry, which makes plasmonic molecules capable of detecting biomolecule clustering and measuring nanometer scale distance fluctuations. In this dissertation, individual plasmonic atoms are firstly evaluated as imaging probe and their interactions with lipid membrane are tested on a newly developed on-chip black lipid membrane system. Subsequently, plasmonic dimers (plasmon rulers) prepared through DNA-programmed self-assembly are monitored to detect the mechanical properties of single biopolymers. Measurement of the spring constant of short (tens of nucleotides or base pairs) DNAs is demonstrated through plasmon coupling microscopy. Colloidal plasmonic atoms of various materials, sizes and shapes scatter vivid colors in the full-visible range. Assembling them into plasmonic molecules provides additional degrees of freedom for color manipulation. More importantly, the electric field in the gaps of plasmonic molecules can be enhanced by several orders of magnitude, which is highly desirable in single molecule sensing applications. In this dissertation, the fundamentals of plasmonic coupling are investigated through one-dimensional gold nanosphere chains. Using the directed self-assembly approach, multichromatic color-switchable plasmonic nanopixels composed of plasmonic atoms and molecules of various materials, sizes, shapes and geometries are integrated in one image with nanometer precision, which facilitates the encoding of complex spectral features with high relevance in security tagging and high density optical data storage.2017-01-01T00:00:00

2차원 유동적 지지형 지질 이중막 마이크로패턴 나노입자 플랫폼을 이용한 DNA 분석

학위논문 (박사)-- 서울대학교 대학원 자연과학대학 화학부, 2017. 8. 남좌민.Lipids are a group of naturally occurring molecules that contain hydrocarbon and are soluble in nonpolar solvents, which are including diverse group of organic compounds such as fats, waxes, steroids, glycolipids, and phospholipids. They play an essential role in organisms such as storing energy, signaling, and constructing cell membrane structures. Phospholipids as the dominant lipid molecules in cell membranes, contain hydrophilic head groups and hydrophobic tails, which governs the spontaneous self-assembled bilayer structure. Then, phospholipid bilayer can be used as a fluidic membrane for the dynamic reaction or a cell mimicking model membrane for the biological study. Especially, supported lipid bilayers (SLBs) of phospholipids on solid glasses can endow the various physical and chemical functionalities of controlling biomolecules reaction, the modification capabilities to incorporate various biomolecules, and the robust platform to monitor the dynamic reactions by optical devices. DNA, deoxyribonucleic acid, is an essential molecule for organisms to survive and reproduce, which contains genetic codes for inheritance. All the genetic information is encoded by a characteristic sequence of four kinds of bases (i.e. adenine (A), cytosine (C), guanine (G), and thymine (T)) in a DNA strand. Mutation or random change in this DNA sequence by accidental exposure to mutagens or copying errors in DNA replication process, can result in the distortion of inheritance or malfunctioned genetic disorder, which cause genetic diseases or cancers. In the past decades, DNA bioassay has received broad attention due to its potential applications in a diversity of fields, e.g., clinical diagnosis, biomedical engineering, food development, environmental protection, forensic investigation and screening of biowarfare agents. One of main challenges in the development of DNA biosensors is the ultrasensitive, quantifiable, and highly reliable DNA detection without any help of amplificationamplification steps using enzymes, fluorescence dyes, and nanomaterials suffer from the erroneous signals by the amplification of the false signals or background signals, need complexed experimental procedures, and cause the signals to be vulnerable to a variety of environmental factors. To realize such an amplification-free, ultrasensitive DNA detection, an endeavor to discriminate rare true-signals from false-signals is necessary. Here, we employed the plasmonic nanoparticles-tethered fluidic SLB platform. Plamonic nanoparticles can generate highly strong light scattering signals and a molecular binding-involved distance change between nanoparticles at the several nanometers level can be detected by the plasmonic coupling. Moreover, the 2D fluidic lipid bilayer can concentrate target DNAs and improve the efficiency of binding reactions in a 2D lipid bilayer pattern. Accordingly, we firstly studied the characteristic properties and fundamental behaviors of SLB layer and nanoparticles on SLB layer with various experimental conditions. Next, the optimized conditions for the ultrasensitive DNA sensing were obtained. Furthermore, this platform and methodology can be applied to the discrimination of various point mutated single-nucleotide polymorphism (SNP) sequences. In chapter 1, we describe recently developed nanomaterial-tethered SLB platform in a formational standpoint. We summarize representative and convenient methods for the formation of supported phospholipid bilayers on planar hydrophilic substrates or micropatterns and linking methods for connecting between nanomaterials and the surface of the bilayer in a synthetic standpoint. We further focus on applications of nanomaterial-tethered SLB in biosensors to detect target molecules with ultrasensitivity and high target specificity. In chapter 2, we studied ultrasensitive and high-selective DNA bioassay through kinetic analysis of dissociation nanodimers using nanomaterial-tethered SLB platform. Amplification/enzyme-free detection and quantification of DNA at ultra-low concentrations, typically 10s-1,000s of targets in solution, is highly challenging but beneficial by offering a more straightforward, less contamination-prone, temperature control-free, less expensive, more quantitative and highly selective detection method than amplification/enzyme-based methods such as the polymerase chain reaction (PCR). Here, we developed an ultrasensitive, highly reliable bio-analytical method [the dynamic analysis on whole nanoparticle cumulative binding events (DANCE)] that allows for quantitatively analyzing dynamically associating and dissociating dimers generated by recognition of DNA strands with two-dimensionally mobile, photostable, and dark-field-detectable DNA-modified nanoparticles (NPs) on a lipid bilayer micropattern. Our results show that the amplification/enzyme-free DANCE provides the PCR-like sensitivity with high target specificity and excellent quantification capability for 10s-1,000s of DNA strands in a whole sample. In chapter 3, we used nanomaterial-tethered SLB biosensor to analyze sensitively mutant position determination of single base mismatched (SBM) DNA sequences. Although the thermodynamic difference among the mutant position-variable SBM sequences is too minuscule to differentiate them, we can measure and analyze the dissociation constants (koff) of various SBM sequences, respectively, which is obtained by counting dissociating nanodimers. As the sequence length is longer, koff value is gradually smaller due to the stability of duplex via multiple Watson-Crick base-pairinghowever, the significant reduction of koff value from 6mer to 7mer sequences were exhibited, which seems to be related to the seven contiguous Watson-Crick base pair rule. By comparison of dissociations among 7mer, 13mer and 15mer DNA duplex systems, we also proved the seven contiguous Watson-Crick base pair rule, even though more than 10mer sequence. Kinetic information of SBM sequences obtained by counting the dissociation events of DNA mediated plasmonic nanodimer, is very sensitive to discriminate mutant point SBM sequences and helpful to understand the mechanism of DNA hybridization dynamics at the single molecular level. Moreover, our research and platform are expected to give much insight into unveiling the dynamic information of various biological reactions among biomaterials such as nucleic acids, proteins, and carbohydrates and the new biological mechanisms in the cellular level.Chapter 1. Introduction: Nanomaterial-tethered Supported Lipid Bilayers and Their Applications 1 1.1 Introduction 2 1.2 Formation of Nanomaterial-tethered Supported Lipid Bilayers (SLB) 5 1.3 Nanomaterial-tethered SLB-Based Bioapplication 15 1.4 Conclusions and perspective 18 1.5 References 19 Chapter 2. Dynamic Analysis on Whole Nanoparticle Cumulative Binding Events for Highly Reliable Detection and Quantification of Trace Amounts of DNA 33 2.1 Introduction 34 2.2 Experimental Section 37 2.3 Results and Discussion 43 2.4 Conclusion 48 2.5 References 49 Chapter 3. The Stability of Single DNA Duplex Monitored on Dissociating Nanodimer Analysis 63 3.1 Introduction 64 3.2 Experimental Section 69 3.3 Results and Discussion 71 3.4 Conclusion 76 3.5 References 78 Abstract in Korean 89Docto

지질 이중층 상 플라즈모닉 나노입자 기반 나노바이오 검지 및 컴퓨팅

학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 화학부, 2019. 2. 남좌민.Supported lipid bilayer is a two-dimensional lipid bilayer self-assembled on a hydrophilic substrate with two-dimensional fluidity. By introducing plasmonic nanoparticles with strong scattering signals into the supported lipid bilayer, it is possible to observe and track thousands of nanoparticles and their interactions at a single-nanoparticle level in real time. In this thesis, I expand the nanoparticle-lipid bilayer platform by engineering plasmonic nanoparticles to construct a complex nanoparticle network system and develop multiplexed bio-detection and bio-computing strategies. Chapter 1 describes a supported lipid bilayer platform incorporating plasmonic nanoparticles. Section 1 introduces the optical properties and biosensing application of plasmonic nanoparticles, and Section 2 introduces tethering technique, characteristics, and advantages for introducing nanoparticles into supported lipid bilayer platforms. In Chapter 2, I introduce a system that can distinguish nine types of nanoparticle assembly reactions occurring simultaneously by introducing optically encoded plasmonic nanoparticles that scatter red, blue, and green light into supported lipid bilayers. I performed multiplexed detection of nine types of microRNAs, which are important gene regulators and cancer cell biomarker. In Chapter 3, I develop a bio-computing platform that recognizes molecular inputs, performs logic circuits, and generates nanoparticle assembly/disassembly output signals. Complex logic circuits are designed and implemented by combining two strategies: (i) interfacial design that constructs a logic circuit through DNA functionalization of the interface of nanoparticles, and (ii) a network design that connects assembly/disassembly reactions. In Chapter 4, I develop a bio-computing calculator capable of performing arithmetic logic operations. I use the nanoparticle-lipid bilayer platform as the hardware that stores, processes, and outputs information, and constructs software that contains logic circuit functions through DNA solution. An information storage nanoparticle stores solution-phase molecular input signals on the surface of nanoparticles. The bio-computing lipid nanotablet recognizes an arithmetic logic circuit programmed with DNA information and generates outputs a result of a kinetic difference between nanoparticle assembly reaction according to the storage state of the input signal.지지형 지질 이중층은 친수성 기판 위에 조립된 2차원의 지질 이중층으로 2차원 상의 유동성을 가진다. 지지형 지질 이중층에 강한 산란 신호를 지니는 플라즈모닉 나노입자를 도입하면 수천 개의 나노입자와 그 상호작용을 단일 나노입자 수준으로 실시간 관찰이 가능하다. 본 학위논문에서는 나노입자-지질 이중층 플랫폼에서의 나노입자 종류 및 개질 방법을 확장하여 복잡한 나노입자 네트워크 시스템을 구성하고, 바이오 검지, 바이오 컴퓨팅 응용을 개발한다. 1장에서는 플라즈모닉 나노입자가 도입된 지지형 지질 이중층 플랫폼을 설명한다. 1절에서 플라즈모닉 나노입자의 광학적 특성과 산란신호를 이용한 바이오센싱 응용 연구를 소개하고 2절에서는 지지형 지질 이중층 플랫폼에 나노입자의 도입 방법, 특징, 장점, 분석방법 등을 소개한다. 2장에서는 빨강, 초록, 파랑 빛을 산란하는 플라즈모닉 나노입자를 합성하고, 지지형 지질 이중층에 도입하여 동시에 일어나는 9종류의 나노입자 결합 반응을 각각 구분할 수 있는 플랫폼을 개발한다. 이를 이용하여 세포 내 중요한 단백질 번역 조절물질이자 암 바이오마커인 마이크로RNA를 동시 다중 검지한다. 3장에서는 지지형 지질 이중층 상에 도입된 나노입자를 다종의 DNA로 기능화하여 특정 DNA 분자 입력 신호 인식, 논리회로 수행, 나노입자 결합/분리 출력 신호 생성하는 바이오 컴퓨팅 플랫폼을 개발한다. 나노입자의 계면을 DNA로 디자인하여 논리 회로를 구성하는 인터페이스 프로그래밍과 나노입자의 결합/분리 반응을 연결하여 네트워크를 디자인하여 논리 회로를 집적하는 네트워크 프로그래밍을 조합하여 복잡한 논리 회로를 설계하고 수행한다. 4장에서는 지지형 지질 이중층에 도입된 나노입자 표면에 용액 상 분자 입력신호를 저장하는 정보 저장 장치를 개발하고 모든 종류의 산술논리연산을 수행할 수 있는 생분자 계산기을 개발한다. 나노입자-지질 이중층 플랫폼을 정보저장, 수행, 출력하는 매체인 하드웨어로 이용하고, DNA 분자 조합 용액을 산술논리회로 기능을 담고있는 소프트웨어로 구성한다. 바이오 컴퓨팅 칩은 DNA 정보로 프로그래밍된 산술논리회로를 인식하여 입력신호의 저장 상태에 따라 나노입자 결합 반응에 반응속도에 차이를 일으키고 결과를 출력한다.Chapter 1. Introduction: Plasmonic Nanoparticle-Tethered Supported Lipid Bilayer Platform 1 1.1. Plasmonic Nanoparticles and Their Bio-Applications 2 1.1.1. Introduction 4 1.1.2. Fundamentals of Plasmonic Nanoparticles 8 1.1.3. Plasmonic Nanoparticle Engineering for Biological Application 11 1.1.4. Plasmonic Nanoparticles for Rayleigh Scattering-Based Biosensing 16 1.1.5. References 21 1. 2. Supported Lipid Bilayer as a Dynamic Platform 24 1.2.1. Introduction 26 1.2.2. Basic Setups and Strategies 29 1.2.3. Nanoparticle-Tethering Techniques 33 1.2.4. Real-Time Imaging and Tracking of Single Nanoparticles on SLB 39 1.2.5. Observation of Interactions between Single Nanoparticles 44 1.2.6. References 50 Chapter 2. Multiplexed Biomolecular Detection Strategy 53 2.1. Introduction 55 2.2. Experimental Section 60 2.3. Results and Discussion 66 2.4. Conclusion 77 2.5. Supporting Information 79 2.6. References 83 Chapter 3. Nano-Bio Computing on Lipid Bilayer 84 3.1. Introduction 85 3.2. Experimental Section 88 3.3. Results and Discussion 98 3.4. Conclusion 120 3.5. Supporting Information 124 3.6. References 161 Chapter 4. Development of Nanoparticle Architecture for Biomolecular Arithmetic Logic Operation 163 4.1. Introduction 165 4.2. Experimental Section 167 4.3. Results and Discussion 171 4.4. Conclusion 177 4.5. References 179 Abstract in Korean 180Docto