항체/항원 상호작용을 감지하는 하이드로겔 기반 SPR 센서 합성

학위논문 (석사) -- 서울대학교 대학원 : 공과대학 화학생물공학부, 2021. 2. 유동원.SPR biosensors are often used for the detection and diagnosis of biomolecular interactions as they allow monitoring of these interactions in real-time, without the need for pre-labelling. However, these SPR biosensors suffer from several limitations such as inability to distinguish between nonspecific and specific interactions and low sensitivities. The sensor chip is one of the most important components of the SPR biosensor and the properties of the sensor chip govern the sensitivity and specificity of the sensor in presence of analyte. Since bioresponsive hydrogels have been reported to have antifouling properties, along with selectivity for specific stimuli, grafting of hydrogels can be expected to improve the specificity of the SPR biosensor. The three-dimensional structure of the gel matrix also provides more attachment sites for ligand immobilization, which can improve the sensitivity of the biosensor. In this work, bioresponsive SPR sensor chips were fabricated from surface modification of bare gold sensor chips with bioresponsive hydrogels. The facile synthesis of hydrogels allowed us to prepare a library of hydrogels with different physical properties. The effect of the physical properties of the hydrogel on the SPR response was then examined. Our results demonstrate the potential of engineering hydrogel properties to produce SPR biosensors of optimal sensitivity in different concentration ranges.SPR 바이오 센서는 사전 라벨링이 필요하지 않은 상태에서 실시간으로 이러한 상호 작용을 모니터링할 수 있기 때문에 생체 분자 상호 작용의 탐지 및 진단 분야에 널리 사용되는 진단 키트이다. 하지만 이러한 SPR 바이오 센서는 비특이적 상호 작용과 특정 상호 작용을 구별할 수 없다는 점과 낮은 민감도와 같은 몇 가지 한계점을 가지고 있다. 센서 칩은 SPR 바이오 센서의 가장 중요한 구성 요소 중 하나이며 센서 칩의 특성은 분석 물질의 존재 하에서 센서의 민감도와 특이성을 좌우한다. 바이오 반응성 하이드로젤은 특정 자극에 대한 선택성과 함께 오손 방지 특성을 가지고 있다고 보고된 바가 있어, 하이드로젤의 이식은 SPR 바이오 센서의 특이성을 향상시킬 것으로 기대된다. 겔 매트릭스의 3차원 구조는 또한 리간드 고정화에 대한 더 많은 부착 부위를 제공하여 바이오 센서의 감도를 향상시킬 수 있다. 본 연구에서는 바이오 반응성 하이드로겔을 이용한 베어 골드 센서 칩의 표면 개질로 바이오 반응성 SPR 센서 칩을 제작하였다. 하이드로겔의 간편한 합성 단계를 통해 우리는 다른 물리적 특성을 가진 하이드로겔 라이브러리를 합성하였다. 또한, 하이드로겔의 물리적 특성이 SPR 반응에 미치는 영향을 조사하였다. 본 연구의 결과는 다양한 농도 범위에서 최적의 감도를 갖는 SPR 바이오센서를 제작하기 위한 하이드로젤의 특성의 가능성을 보여준다.Chapter 1. Introduction 1 1.1 Surface plasmon resonance biosensors 1 1.2 Use of hydrogels for SPR biosensing 3 Chapter 2. Experimental Methods 6 2.1 Materials 6 2.2 Hydrogel synthesis 7 2.3 Characterization of prepared hydrogels 10 2.4 PD-1 receptor conjugation to hydrogels 10 2.5 Analyte detection through SPR analysis 11 Chapter 3. Results and Discussion 13 3.1 Precipitation polymerization of hydrogels 13 3.2 Characterization of hydrogels 16 3.3 Influence of crosslinker concentration on hydrogel size 19 3.4 Influence of surfactant on hydrogel synthesis 21 3.5 Influence of initiator concentration on hydrogel size 22 3.6 Influence of polymerization temperature on size 23 3.7 SPR sensing of PD-1Ab 27 3.8 Effect of hydrogel size on SPR response 29 3.9 Effect of crosslinking density on SPR response 33 Chapter 4. Conclusion 36 References 38 Abstract in Korean 46Maste

Constructing The Working Model of A 2-Up System Pyramid Scheme

A pyramid scheme is a business model that is illegal in many countries. It works by recruiting members via promises of payments or services by enrolling more people in the scheme. In recent years, pyramid schemes have been expanding, and many people have fallen victim when the schemes collapse. In this study, the working model and formulation of the 2-up system pyramid scheme are studied to identify its fundamental elements and working mechanism. A model is constructed mathematically to visualise the system’s profitability. To further prove that a pyramid scheme is unsustainable, the limitations of a 2-up system may lead to the collapse of the pyramid structure, and a simulation study on the probability of an individual losing money in a 2-up system is investigated. The method of investigation involved literature reviews and data visualisation. As no references are available on the 2-up system formula, the reading of research articles and websites became the primary source of study. Data visualisation is carried out to showcase the cash flow of profits in a 2-up system pyramid scheme. The fundamental element of the system is for one member to recruit at least three others to prevent a loss as profits from the first two recruits will be channelled to an upline member. According to the investigation of limitations, approximately 65.78% of the world population would need to join the 2-up system when the level rises to 21, where the pyramid structure is vulnerable to collapse. There is a possibility of pyramid scheme members suffering losses since there is a requirement to make a profit. Pyramid schemes are destined to fail in the long run as the possible returns of an individual is never guarantee

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Label-Free Analysis of Multivalent Protein Binding Using Bioresponsive Nanogels and Surface Plasmon Resonance (SPR)

Precise identification of protein-protein interactions is required to improve our understanding of biochemical pathways for biology and medicine. In physiology, how proteins interact with other proteins or small molecules is crucial for maintaining biological functions. For instance, multivalent protein binding (MPB), in which a ligand concurrently interacts with two or more receptors, plays a key role in regulating complex but accurate biological functions, and its interference is related to many diseases. Therefore, determining MPB and its kinetics has long been sought, which currently requires complicated procedures and instruments to distinguish multivalent binding from monovalent binding. Here, we show a method for quickly evaluating the MPB over monovalent binding and its kinetic parameters in a label-free manner. Engaging pNIPAm-co-AAc nanogels with MPB-capable moieties (e.g., PD-1 antigen and biocytin) permits a surface plasmon resonance (SPR) instrument to evaluate the MPB events by amplifying signals from the specific target molecules. Using our MPB-based method, PD-1 antibody that forms a type of MPB by complexing with two PD-1 proteins, which are currently used for cancer immunotherapy, is detectable down to a level of 10 nM. In addition, small multivalent cations (e.g., Ca2+, Fe2+, and Fe3+) are distinguishably measurable over monovalent cations (e.g., Na+ and K+) with the pNIPAm-co-AAc nanogels.N

Green extraction of healthy and additive free mitochondria with a conventional centrifuge

In this research, we propose a novel centrifugal device for the massive extraction of healthy mitochondria with a centrifuge used in general laboratories within 30 minutes. The device mainly consists of two key components. One component is a microfluidic device, which is fabricated by photolithography, nickel electroforming, and polydimethylsiloxane casting, for the efficient disruption of the cell membrane. The other component is a stainless steel container, which is manufactured by computer numerical control machining, for the storage of the cell suspension. After assembly, the appropriate number of cells is pushed through the microfluidic device for cell membrane disruption by centrifugal force generated by a general laboratory centrifuge. The solution which contains cell debris and mitochondria are collected to purify the crude mitochondria via differential centrifugation. Compared with the quantity and efficiency of mitochondria isolated from the same number of cells using a conventional kit, device-extracted mitochondria show a more complete mitochondrial electron transport chain complex and a similar number of mitochondria verified by Western blot analysis of mitochondrial complexes I–V and mitochondrial outer membrane protein Tom20, respectively, as well as a normal mitochondrial structure revealed by transmission electron microscopy. Moreover, the mitochondrial membrane potential of device-extracted mitochondria stained with tetramethylrhodamine ethyl ester is higher than that of kit-extracted mitochondria. Furthermore, the coculture of device-extracted mitochondria with fibroblasts revealed that fibroblasts could uptake foreign mitochondria through endocytosis without drug treatment. These results show that the proposed microfluidic device preserves mitochondrial protein structure, membrane integrity, and membrane potential within 30 minutes of extraction and is a useful tool for therapeutic mitochondrial transplantation and regenerative medicine

Detection of α‑Thrombin with Platelet Glycoprotein Ibα (GP1bα) for the Development of a Coagulation Marker

The detection of prothrombotic markers is crucial for understanding thromboembolism and assessing the effectiveness of anticoagulant drugs. α-Thrombin is a marker that plays a critical role in the coagulation cascade process. However, the detection of this enzymatic molecule was hindered by the absence of an efficient modality in the clinical environment. Previously, we reported that one α-thrombin interacts with two α-chains of glycoprotein Ib (GPIbα), i.e., multivalent protein binding (MPB), using bioresponsive hydrogel nanoparticles (nanogels) and optical microscopy. In this study, we demonstrated that GPIbα-mediated platforms led to the highly sensitive and quantitative detection of α-thrombin in various diagnostic systems. Initially, a bioresponsive nanogel-based surface plasmon resonance (nSPR) assay was developed that responds to the MPB of α-thrombin to GPIbα. The use of GPIbα for the detection of α-thrombin was further validated using the enzyme-linked immunosorbent assay, which is a gold-standard protein detection technique. Additionally, GPIbα-functionalized latex beads were developed to perform latex agglutination (LA) assays, which are widely used with hospital diagnostic instruments. Notably, the nSPR and LA assays exhibited a nearly 1000-fold improvement in sensitivity for α-thrombin detection compared to our previous optical microscopy method. The superiority of our GPIbα-mediated platforms lies in their stability for α-thrombin detection through protein–protein interactions. By contrast, assays relying on α-thrombin enzymatic activity using substrates face the challenge of a rapid decrease in postsample collection. These results suggested that the MPB of α-thrombin to GPIbα is an ideal mode for clinical α-thrombin detection, particularly in outpatient settings

Simple Host-Guest Assembly for High-Resolution Magnetic Resonance Imaging of Microvasculature

© Magnetic resonance angiography (MRA) is an important imaging technique that can be used to identify and characterize various types of vascular diseases. However, currently used molecular contrast agents are unsuitable for MRA due to the short intravascular retention time, the whole-body distribution, and the relatively low contrast effect. In this study, we developed a vascular analysis contrast agent (i.e., VasCA) for MRA, which is a simple and biocompatible 1:1 host-guest assembly of PEGylated β-cyclodextrin and gadolinium chelate with renal clearable size and high relaxivity (r1 = 9.27 mM-1 s-1). Its biocompatibility was confirmed by in vivo animal studies as well as in vitro 3D cell culture. In a tumor-bearing rat model, VasCA circulated in the blood vessels much longer (4.3-fold increase) than gadoterate meglumine (Dotarem) and was mainly excreted by the renal system after intravenous injection. This feature of VasCA allows characterization of tumor microvasculature (e.g., feeding and draining vessels) as well as visualization of small vessels in the brain and body organs. Furthermore, after treatment with an angiogenesis inhibitor (i.e., sorafenib), VasCA revealed the vessel normalization process and allowed the assessment of viable and necrotic tumor regions. Our study provides a useful tool for diverse MRA applications, including tumor characterization, early-stage evaluation of drug efficacy, and treatment planning, as well as diagnosis of cardiovascular diseases.11Nsciescopu

Tuning Surface Plasmon Resonance Responses through Size and Crosslinking Control of Multivalent Protein Binding-Capable Nanoscale Hydrogels

© 2022 American Chemical Society.Surface plasmon resonance (SPR) phenomena have been widely studied to detect biomolecules because of their high sensitivity and ability to determine biomolecular interactions with kinetic information. However, highly selective detection in specific concentration ranges relevant to target biomolecules is still a challenging task. Recently, we developed bioresponsive nanoscale hydrogels to selectively intensify SPR signals through multivalent protein binding (MPB) events with target biomolecules, including IL-2, where we were able to demonstrate exceptional selectivity for target biomolecules with minimal responses to nonspecific and monovalent binding events. In this work, we systematically explored the relationship between the physical properties of MPB-capable nanoscale hydrogels and their SPR response induced in the presence of the programmed cell death protein 1 antibody (PD-1Ab) as a model target biomolecule. First, we developed a synthetic protocol by controlling various reaction parameters to construct a library of nanoscale poly(N-isopropylacrylamide-co-acrylic acid) hydrogels (NHs) with different sizes (from 400 nm to 1 μm) and degrees of crosslinking (from 2 to 8%). Then, by incorporating MPB-capable PD-1 receptors onto the surface of NHs to form PD-1-responsive nanoscale hydrogels (PNHs), the hydrogel size and crosslinking dependency of their SPR responses were investigated. Our results reveal the appropriate hydrogel size regime and degree of crosslinking for effective PD-1Ab detection at specific concentrations range between a few nM and 1 μM. Overall, our study demonstrates that by tuning the physical properties of the nanoscale hydrogel matrix, the sensitivity and detection range of MPB-based SPR sensors can be modulated to potentially benefit clinical applications such as monitoring diverse therapeutic biomolecules.11Nsciescopu