수용체의 작용을 연구하기 위한 생체분자-탄소나노튜브 융합 구조 및 바이오전자 센서로의 응용

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 물리·천문학부(물리학전공), 2022.2. 홍승훈.Living organisms can perceive various external materials such as pathogens and tastants through sensory receptors. Various biosensors using these receptors have been developed for the monitoring of receptor activity and the detection of various molecules. Conventional methods using receptors include receptor-expressed cell-based devices or membrane receptor-based sensors. Recently, new types of nanostructures such as nanovesicles and nanodiscs have been introduced. However, these novel constructs have limitations such as low receptor expression yield and complicated production process. In this dissertation, the development and application of biosensors using new types of bioreceptors will be discussed. First, it will be discussed about the bioelectronic tongue devices using a ligand-binding domain of the human sweet taste receptor to monitor the activities of the receptor. In this work, only the VFT domain of the human sweet taste receptor was expressed. The VFT domain was immobilized on the floating electrodes of carbon nanotube field-effect transistors. We demonstrated the monitoring of the response of the receptor to the sweet taste substance using the device in real-time. In addition, by using this sensor, it was possible to analyze the enhancement and inhibition effect of sweet taste by various substances. Furthermore, it could be used to detect saccharides in real beverage samples. The sensor has the advantage of reusability and long-term storability. Our VFT domain-based bioelectronic tongue can be a powerful tool for the detection of sweet taste substances in the food and healthcare industry. Next, the monitoring of immune cell responses and their applications using antibody-inoculated immune cell-derived nanovesicles will be discussed. Nanovesicles were extracted from RBL-2H3 cells, and they were inoculated with IgE antibodies for the binding of target antigens. The nanovesicles were combined with carbon nanotubes field-effect transistors to fabricate an allergen biosensor. This biosensor can detect antigens in real-time, and it also enables the detection of allergens in real food. In addition, drug effects on the nanovesicle were evaluated. Importantly, we can easily obtain the antibody-inoculated nanovesicles for target antigens by inoculating antibodies. Therefore, our system can be an adaptive and universal platform for the detection and monitoring of various antigens.살아있는 유기체는 감각 수용체를 사용하는 감각 시스템을 통해 병원체 및 미각 물질과 같은 다양한 외부 변화를 감지할 수 있습니다. 이러한 수용체를 활용한 다양한 바이오센서들이 활용되어오고 있다. 기존의 수용체 활용 방식으로는 수용체가 발현된 세포를 활용하거나, 막단백질 자체를 이용하는 방법들이 일반적이다. 최근에는 수용체가 발현된 나노베지클이나 나노디스크 같은 새로운 종류의 나노구조체들이 개발되었다. 하지만 이러한 새로운 구조체들도 낮은 수용체의 발현율, 복잡한 제조 방법 등의 한계점이 있다. 이 학위논문에서는, 이러한 수용체 기반 나노구조체들의 단점을 보완하기 위한 새로운 구조의 수용체 기반 바이오리셉터들을 활용한 바이오센서 개발과 그 활용에 대해 논의한다. 먼저, 인간의 단맛 수용체의 작용을 모니터링하기 위한 수용체의 일부 도메인을 이용한 바이오 전자혀 센서에 대해 논의할 것이다. 이를 위하여, 인간 단맛 수용체에서 VFT 도메인만을 발현시켰다. VFT 도메인을 부유 전극이 있는 탄소나노튜브 전계 효과 트랜지스터에 결합시켜 단맛 감지 센서로 제작하였다. 이를 통하여, 단맛 물질에 대한 단맛 수용체의 반응을 실시간으로 모니터링할 수 있었다. 또한, 이 센서를 활용하여 다양한 물질에 의한 단맛의 향상 및 억제 효과를 분석할 수 있었다. 그리고 실제 음료에 존재하는 당류를 검출하는데도 활용할 수 있었다. 이 센서는 여러 번 재사용할 수 있으며, 오랜 기간 보관할 수 있는 장점이 있다. 다음으로, 면역세포 유래 나노베지클에 항체를 접종한 새로운 종류의 바이오리셉터를 활용한 면역 세포 반응 모니터링 및 그 활용에 대해 논의할 것이다. 면역 세포에서 추출한 나노베지클에 항체를 접종하여 다양한 항원에 결합할 수 있는 바이오리셉터를 제작했다. 이를 탄소나노튜브에 결합하여 항원 감지 바이오 센서로 제작하였다. 이 바이오센서는 실시간으로 항원의 검출이 가능하며, 실제 음식에 들어있는 알러젠 검출도 가능하다. 또한, 약물에 의한 면역 세포 기능 변화를 평가할 수 있다. 더욱이, 항체가 접종된 나노베지클은 항체만 바꿔주면 타겟 항원에 결합하는 구조를 쉽게 얻을 수 있어 다양한 항원 검출에 범용적으로 활용할 수 있다.Chapter 1. Introduction 1 1.1. Membrane Receptor Protein 2 1.2. Methods for the Monitoring of Receptor Activity 4 1.3. Carbon Nanotube Field-Effect Transistor 6 Chapter 2. Ultrasensitive Bioelectronic Tongue Based on Ligand Binding Domain of Sweet Taste Receptor for the Monitoring of Sweet Taste Receptor 8 2.1. Introduction 9 2.2. Fabrication of a Floating Electrode-Based Sweet Taste Sensor and Preparation of Samples 13 2.3. Characteristics of a Sweet Taste Sensor 16 2.4. Detection of Sweet Tastants by Using Sweet Taste Sensor 19 2.5. Evaluation of Sweet Substances in Real Beverages 24 2.6. Inhibition and Enhancement of Sweet Taste 27 2.7. Summary 30 Chapter 3. Nanovesicle-Based Biosensor for Monitoring of Immune Cell in Allergic Response 31 3.1. Introduction 32 3.2. Fabrication of an Immune Cell-Derived Sensor and Preparation of Samples 34 3.3. Characterization of Nanodisc-Immobilized CNT-FET 38 3.4. Monitoring of the Responses of Immune Cell-Derived Nanovesicles 40 3.5. Detection of Allergens in Real Food Samples 43 3.6. Summary 45 Conclusion 46 Bibliography 48 Abstract in Korean 56박

Dual regulation of R-type CaV2.3 current by Gq-coupled receptors

Many high voltage-activated Ca2+ channels are modulated by Gq-coupled M1 muscarinic acetylcholine receptors. CaV2.3 currents are known to be increased by M1 receptor activation, and the increase in the CaV2.3 currents is mediated by phosphorylation of CaV2.3 channel via the activation of protein kinase C (PKC). Here, we report that M1 muscarinic receptors can also inhibit CaV2.3 currents when the channels are fully activated by PKC. In the whole-cell configuration of tsA201 cells, phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated CaV2.3 currents by ~ 2-fold. We found that after the PMA-induced potentiation of CaV2.3 currents, application of the M1 receptor agonist oxotremorine-M (Oxo-M), decreased the currents by 52%. We examined if the hydrolysis of plasma membrane phosphoinositides (PIs) were involved in the muscarinic suppression of CaV2.3 currents. We used two methods to deplete PI(4,5)P2; voltage-sensing phosphatase (VSP), and rapamycin-induced translocatable pseudojanin (PJ) system. Activation of VSP suppressed CaV2.3 current by 38%. PJ system could directly dephosphorylate 4- and 5-phosphates from both PI(4)P and PI(4,5)P2 the plasma membrane. After the addition of rapamycin CaV2.3 currents were dramatically and irreversibly decreased by 66% compared to the initial level. Taken together, our results suggest that CaV2.3 currents are modulated by M1 receptor in a dual mode; potentiation by PKC activation and suppression by poly-PI depletion. Activation of M1 receptors can solely decrease CaV2.3 currents in the PKC-activated cells. PJ-induced inhibition of CaV2.3 currents demonstrates that poly-PIs are important in the maintenance of CaV2.3 channel activity. ⓒ 2015 DGIST1. Introduction 1 -- 2. Materials and methods 4 -- 2.1 Materials 4 -- 2.2 Cell culture 4 -- 2.3 Transfection 5 -- 2.4 Solution 5 -- 2.5 Chemicals 6 -- 2.6 Current recording 6 -- 2.7 Confocal imaging 6 -- 2.8 Data analysis 7 -- 3. Results 8 -- 3.1 CaV2.3 currents are suppressed as well as stimulated by M1 muscarinic receptor 8 -- 3.2 CaV2.3 currents are decreased by Dr-VSP activation 9 -- 3.3 CaV2.3 currents are decreased by chemically-induced phosphoinositide depletion 9 -- 4. Discussion 12 -- 5. Figure legends 15 -- 6. Figures 19 -- References 32 -- Abstract in Korean 36많은 전압 개폐 칼슘 채널은 Gq 단백질 연결 수용체 (Gq-protein coupled receptor, GqPCR) 중 하나인 무스카린성 아세틸콜린 수용체에 의해서 조절된다. 전압 개폐 칼슘 채널 중 CaV2.3 채널의 전류는 M1 무스카린성 수용체에 의해서 증가하며 이 때 전류의 증가는 단백질 인산화 효소 C에 의한 채널의 인산화 때문이라고 알려져 있다. 실제로 CaV2.3 채널을 발현하는 tsA201 세포에 phorbol 12-myristate 13-acetate (PMA) 라는 단백질 인산화 효소 C의 활성제를 처리할 경우 CaV2.3 채널의 전류가 두 배 가량 증가하는 것을 보았다. 흥미로운 점은 PMA 를 처리해서 CaV2.3 채널을 완전히 활성화 시킨 후 M1 수용체를 활성화 시키면 CaV2.3 채널의 전류가 줄어든다는 점이다. 우리는 전류를 억제시키는 요인을 찾기 위해서 M1 수용체에 의해 일어나는 신호전달계를 살펴보았다. M1 수용체가 활성화 되면 세포막에 있는 PI(4,5)P2 라는 인지질이 분해된다. CaV2.3 채널과 같은 그룹인 CaV2.2 채널이 PI(4,5)P2 양이 감소하면 전류가 줄어든다는 보고가 있기 때문에 PI(4,5)P2 가 CaV2.3 채널의 억제에도 영향을 미치는지 알아보기로 했다. 첫 번째로 높은 전압 (+120 mV)을 가해줬을 때 활성화되는 인산 가수분해 효소를 이용했다. 우리는 이 방법을 통해서 CaV2.3 채널의 전류가 38% 가량 줄어든다는 것을 알아냈다. 두 번째로 특정 화학물질을 첨가하였을 때 두 개의 분자가 이합체화 되는 현상을 이용하는 방법을 사용했다. 이 방법을 이용해서 인산 가수분해 효소를 세포막으로 가져올 수 있고 인지질을 분해 할 수 있다. 우리는 라파마이신이라는 화학물질을 이용해서 인산 가수분해 효소를 세포막으로 이동시킨 후 PI(4,5)P2의 양을 감소시켰다. 그 결과 CaV2.3 채널의 전류가 66% 정도 줄어드는 것을 발견했다. 위의 실험결과들은 CaV2.3 채널의 전류가 M1 수용체에 의해서 활성화 될 수도 있고 억제될 수도 있다는 것을 보여준다. 활성화되는 기작은 단백질 인산화 효소 C에의한 CaV2.3 채널의 인산화 때문이고 억제되는 기전은 세포막에 있는 PI(4,5)P2 의 감소에 의한 것이다. 이 연구를 통해서 세포막에 있는 PI(4,5)P2는 CaV2.3 채널의 활성을 유지하고 조절하는데 중요한 역할을 한다는 것을 밝혀냈다. ⓒ 2015 DGISTMasterdCollectio

Mirage cosmology with an unstable probe D3-brane

We consider the mirage cosmology by an unstable probe brane whose action is represented by BDI action with tachyon. We study how the presence of tachyon affects the evolution of the brane inflation. At the early stage of the brane inflation, the tachyon kinetic term can play an important role in curing the superluminal expansion in mirage cosmology.Comment: 11 pages, improved presentation with some clarifications, typos corrected, references adde

Targeting the insulin growth factor-1 receptor with fluorescent antibodies enables high resolution imaging of human pancreatic cancer in orthotopic mouse models.

The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of pancreatic cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24-31) was conjugated with 550 nm or 650 nm fluorophores. Western blotting confirmed the expression of IGF-1R in Panc-1, BxPC3, and MIAPaCa-2 human pancreatic cancer cell lines. Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of the pancreatic cancer cells. Subcutaneous Panc-1, BxPC-3, and MIA PaCa-2 tumors became fluorescent after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted BxPC-3 tumors became fluorescent with the conjugated IGF-1R antibodies, and were easily visible with intravital imaging. Gross and microscopic ex vivo imaging of resected pancreatic tumor and normal pancreas confirmed that fluorescence indeed came from the membrane of cancer cells, and it was stronger from the tumor than the normal tissue. The present study demonstrates that fluorophore-conjugated IGF-1R antibodies can visualize pancreatic cancer and it can be used with various imaging devices such as endoscopy and laparoscopy for diagnosis and fluorescence-guided surgery

Emotional Landscapes

“Emotional Landscape” delivers a sense of gravity, openness, and breathing space through oil paintings on linen of abstracted bodily forms. The imagery in the works generates an atmosphere where one can feel rooted and anxiety-free. The paintings invite a close read of the complexities of compounded affects

Fluorescent-Antibody Targeting of Insulin-Like Growth Factor-1 Receptor Visualizes Metastatic Human Colon Cancer in Orthotopic Mouse Models.

Fluorescent-antibody targeting of metastatic cancer has been demonstrated by our laboratory to enable tumor visualization and effective fluorescence-guided surgery. The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of metastatic colon cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24-31) was conjugated with 550 nm, 650 nm or PEGylated 650 nm fluorophores. Subcutaneous, orthotopic, and liver metastasis models of colon cancer in nude mice were targeted with the fluorescent IGF-1R antibodies. Western blotting confirmed the expression of IGF-1R in HT-29 and HCT 116 human colon cancer cell lines, both expressing green fluorescent protein (GFP). Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery