Bioelectronic Noses Based on Olfactory Receptors

Non

나노디스크 및 나노베지클에 내장된 후각 수용체의 최적의 생산과 냄새의 패턴 및 시각화에 대한 응용

학위논문(박사) -- 서울대학교대학원 : 공과대학 화학생물공학부, 2022. 8. 박태현.G protein-coupled receptors (GPCRs) are the most intensively studied for screening drug targets. Especially, class A GPCR including olfactory receptor (OR) which accounts for about 85 % of GPCR family is more important for codifying and screening target receptors. There are about 400 kinds of ORs in human olfactory system. The interactions between ORs and odorants generate signals which are transferred to brain as combinatorial codes. Humans can discriminate more than 1 trillion olfactory stimuli with a limited number of ORs because of widespread OR-driven modulation such as inhibition and enhancement in peripheral olfactory coding. Since the sense of smell perceives the complex external world as a pattern, many studies have been conducted to mimic the response of ORs. In particular, protein-based nanobiosensor is expected as a platform to mimic the olfaction because it has advantages such as mass production, ease of reuse, and low cost. However, reconstitution of the structure of GPCRs is challenging because almost all GPCRs produced in E. coli system are expressed as inclusion bodies. For this reason, reconstitution techniques have been developed to recover the functionality of GPCRs, such as the use of detergent micelles, nanovesicles, bicelles and nanodiscs (NDs). Among these materials, NDs have been considered the most effective reconstitution material because of their stability in various environments and their functional lifetimes. In this thesis, ORs were produced in E. coli system with high productivity and reconstituted to ND or nanovesicle forms. Then the functional reconstituted ORs were applied to monitoring meat freshness/spoilage, disease diagnosis and practical colorimetric sensor. First, OR was overexpressed by coexpressing effector genes, such as djlA, the membrane-bound DnaK cochaperone, and rraA, inhibitor of the mRNA-degrading activity of E. coli RNase E. The E. coli strains coexpressing DjlA or RraA suppressed protein-induced toxicity and overexpressed the ORs. By controlling the molar ratio of OR, membrane scaffold protein, and phospholipid, ND of appropriate size were made, and high-purity ND could be purified. OR-embedded NDs showed stability to various temperature and storage time. Second, Human ORs which bind to gastric cancer and halitosis biomarkers were successfully reconstituted to ND form and purified. The NDs had various patterns to artificial saliva samples because NDs had various binding affinities to target molecules. Through principal component analysis of various patterns for artificial saliva samples, it was possible to distinguish between healthy control samples and patient samples. Third, trace amine-associated receptors (TAARs), TAAR13c and TAAR13d, were successfully overexpressed in E. coli system and reconstituted to ND form. These NDs were utilized for development of ND-based BE-nose for monitoring meat freshness. The ND-based BE-noses was successfully performed towards diverse on-site and the various real samples and could be used to monitor freshness of meat. Lastly, human OR1A2 (hOR1A2) was reconstituted into detergent micelle and it was used for development of colorimetric sensor detecting geraniol. Polydiacetylene (PDA) was used as secondary transducer for visualization of responses of OR. The structural and functional properties of the hOR1A2 were maintained when it was embedded in PDA/lipid nanovesicles. The hOR1A2 embedded in PDA/lipid nanovesicle caused a color transition from blue to purple when it reacted with geraniol, whereas there was no color transition when it reacted with other molecules. In this study, various ORs were successfully reconstituted with ND or nanovesicles. The reconstructed OR is expected to be applied to food freshness monitoring, disease diagnosis by pattern analysis, and practical colorimetric sensors.G 단백질 연결 수용체 (GPCR)는 약물 표적 스크리닝을 위해 가장 집중적으로 연구되는 단백질이다. 특히, GPCR 계열의 약 85%를 차지하는 후각 수용체 (OR)를 포함하는 클래스 A GPCR은 표적 수용체를 코드화하고 스크리닝하는 데 더 중요하다. 인간의 후각 시스템에는 약 400 종류의 OR이 있다. OR과 냄새 물질 사이의 상호 작용은 패턴의 조합으로 뇌에 전달되는 신호를 생성한다. 인간은 말초 후각 코딩의 억제 및 향상과 같은 광범위한 OR에 의한 조절로 인해 제한된 수의 OR로도 1조개 이상의 후각 신호를 구별할 수 있다. 후각은 복잡한 외부 세계를 패턴으로 인식하기 때문에 OR의 반응을 모방하기 위해 많은 연구가 수행되었다. 특히, 단백질 기반 나노바이오센서는 양산성, 재사용 용이성, 저비용 등의 장점이 있어 후각을 모방하는 플랫폼으로 기대된다. 그러나 GPCR 구조의 재구성은 대장균 시스템에서 생성되는 거의 모든 GPCR이 봉입체로 발현되기 때문에 어려운 일이다. 이러한 이유로 세제 미셀, 나노베지클, 바이셀 및 나노디스크 (ND)와 같은 GPCR의 기능을 복구하기 위한 재구성 기술이 개발되었다. 이들 물질 중 ND는 다양한 환경에서의 안정성과 기능적 수명 때문에 가장 효과적인 재구성 물질로 여겨져 왔다. 본 논문에서는 대장균 시스템에서 높은 생산성으로 OR을 생산하여 나노디스크 또는 나노베지클 형태로 구조를 재구성하였다. 그런 다음 기능적으로 재구성된 OR을 육류 신선도/부패 모니터링, 질병 진단 및 실용적인 비색 센서에 적용했다. 첫째, OR은 djlA, 막 결합 DnaK cochaperone 및 rraA, E. coli RNase E의 mRNA 분해 활성 억제제와 같은 이펙터 유전자를 공동 발현함으로써 과발현되었다. DjlA 또는 RraA를 공동 발현하는 대장균 균주는 단백질 발현에 의한 독성을 억제하고 후각 수용체를 과발현했습니다. 후각 수용체, 막 지지체 단백질, 인지질의 몰비를 조절하여 적절한 크기의 ND를 만들고 고순도 ND를 정제할 수 있었다. 후각 수용체가 내장된 ND는 다양한 온도 및 보관 시간에 대해 안정성을 보였다. 둘째, 위암 및 구취 바이오마커에 결합하는 인간 OR은 ND 형태로 성공적으로 재구성되고 정제되었다. ND는 표적 분자에 대한 다양한 결합 친화성을 가졌기 때문에 인공 타액 샘플에 대한 다양한 패턴을 가졌다. 인공 타액 샘플에 대한 다양한 패턴의 주성분 분석을 통해 건강한 대조군 샘플과 환자 샘플을 구별할 수 있었다. 셋째, 미량 아민 관련 수용체 (TAAR), TAAR13c 및 TAAR13d가 대장균 시스템에서 성공적으로 과발현되었고 ND 형태로 재구성되었다. 이러한 ND는 육류 신선도를 모니터링하기 위한 ND 기반 전자 코의 개발에 활용되었다. ND 기반 전자 코는 다양한 현장 및 실제 샘플에 성공적으로 작동되었으며 육류의 신선도를 모니터링하는 데 사용할 수 있었다. 마지막으로 인간 OR1A2 (hOR1A2)를 세제 미셀로 재구성하여 제라니올을 감지하는 비색 센서 개발에 활용하였다. 폴리다이아세틸렌 (PDA)은 후각 수용체의 반응을 시각화하기 위한 2차 변환기로 사용되었다. hOR1A2의 구조적 및 기능적 특성은 PDA/지질 나노베지클에 내장되었을 때 유지되었다. PDA/지질 나노베지클에 내장된 hOR1A2가 geraniol과 반응할 때 파란색에서 보라색으로 색상 전이를 일으킨 반면 다른 분자와 반응할 때는 색상 전이가 없었습니다. 이 연구에서는 다양한 OR이 ND 또는 나노베지클로 성공적으로 재구성되었다. 재구성된 OR은 식품 신선도 모니터링, 패턴 분석에 의한 질병 진단 및 실용적인 비색 센서에 적용될 수 있을 것으로 기대가 된다.Chapter 1 Research background and objective 15 Chapter 2 Literature review 20 2.1 Olfaction 22 2.1.1 Olfactory system 21 2.1.2 Olfactory receptors 25 2.1.3 Patterns of odorants 29 2.2 Nanobiosensor 32 2.2.1 Nanobiosensor system 32 2.2.2 Components for the nanobiosensor 35 2.2.3 Nanobiosensor detecting smell 37 2.3 Biomarkers in disease and food spoilage 44 2.3.1 Gastric cancer 44 2.3.2 Halitosis 47 2.3.3 Meat spoilage 49 2.4 Expression of GPCR in E. coli system 50 Chapter 3 Experimental procedures 52 3.1 Materials 53 3.2 Gene Cloning . 54 3.3 Expression 54 3.3.1 Expression of olfactory receptors in E. coli. 54 3.3.2 Expression of membrane scaffold protein in E. coli 56 3.3.3 Expression of olfactory receptors in HEK-293T cell 57 3.4 Purification. 57 3.4.1 Purification of olfactory receptors. 57 3.4.2 Purification of membrane scaffold protein 58 3.5 Functional reconstitution of olfactory receptors 59 3.5.1 Nanodisc 59 3.5.2 Detergent micelle. 60 3.5.3 Polydiacetylene/Lipid nanovesicle 61 3.6 Characterization . 61 3.6.1 Nano-glo dual luciferase assay 61 3.6.2 SDS-PAGE analysis 62 3.6.3 Dynamic light scattering 63 3.6.4 Circular dichroism . 63 3.6.5 Tryptophan fluorescence quenching assay 63 3.7 Immobilization of olfactory receptor-embedded nanodisc on graphene 64 Chapter 4 Enhancement of olfactory receptor production in E. coli system and characterization of olfactory receptor-embedded nanodiscs. 66 4.1 Introduction 67 4.2 Expression and purification of olfactory receptor in E. coli system 69 4.3 Purification and size analysis of olfactory receptor-embedded nanodiscs. 73 4.4 Stability of immobilized olfactory receptor-embedded nanodiscs. 77 4.5 Conclusions 82 Chapter 5 Development of nanodisc-based bioelectronic nose using trace amine-associated receptors for monitoring meat freshness/spoilage . 84 5.1 Introduction 85 5.2 Characterization of TAARs 87 5.3 Performance of nanodisc-based bioelectronic nose in the liquid phase. 91 5.4 Gas sensing performance of bioelectronic nose and its application to meat spoilage 97 5.5 Measurements of real samples using TAAR nanodisc-based bioelectronic nose 99 5.6 Conclusions 104 Chapter 6 Pattern analysis for gastric cancer biomarkers using human olfactory receptor-embedded nanodiscs . 105 6.1 Introduction 106 6.2 Affinities of human olfactory receptor-embedded nanodiscs to gastric cancer biomarkers 108 6.3 Patterns for gastric cancer biomarkers in artificial saliva 112 6.4 Principal component analysis for artificial saliva samples 115 6.5 Conclusions 117 Chapter 7 Pattern analysis for halitosis biomarkers in artificial saliva using olfactory receptor-embedded nanodiscs 118 7.1 Introduction 119 7.2 Characterization of olfactory receptor-embedded nanodiscs. 120 7.3 Patterns for halitosis biomarkers in artificial saliva. 122 7.4 Principal component analysis for artificial saliva samples 125 7.5 Conclusions 127 Chapter 8 Visual detection of geraniol using human olfactory receptor embedded in polydiacetylene/lipid nanovesicle 128 8.1 Introduction 129 8.2 Functionality of hOR1A2 embedded in detergent micelle and PDA/lipid nanovesicle. 130 8.3 Structural assay of hOR1A2 embedded in detergent micelle and PDA/lipid nanovesicle. 133 8.4 Size analysis and morphology of hOR1A2 embedded in PDA/lipid nanovesicle. 135 8.5 Photoluminescence intensity of hOR1A2 embedded in PDA/lipid nanovesicle. 137 8.6 Conclusions 141 Chapter 9 Overall discussion and further suggestions. 142 Bibliography . 147 국문초록 157박

Nanobiosensors based on individual olfactory receptors

In the SPOT-NOSED European project, nanoscale sensing elements bearing olfactory receptors and grafted onto functionalized gold substrates are used as odorant detectors to develop a new concept of nanobioelectronic nose, through sensitive impedancemetric measurement of single receptor conformational change upon ligand binding, with a better specificity and lower detection threshold than traditional physical sensors.Comment: Submitted on behalf of EDA Publishing Association (http://irevues.inist.fr/EDA-Publishing

Olfactory receptors for a smell sensor: A comparative study of the electrical responses of rat I7 and human 17-40

In this paper we explore relevant electrical properties of two olfactory receptors (ORs), one from rat OR I7 and the other from human OR 17-40, which are of interest for the realization of smell nanobiosensors. The investigation compares existing experiments, coming from electrochemical impedance spectroscopy, with the theoretical expectations obtained from an impedance network protein analogue, recently developed. The changes in the response due to the sensing action of the proteins are correlated with the conformational change undergone by the single protein. The satisfactory agreement between theory and experiments points to a promising development of a new class of nanobiosensors based on the electrical properties of sensing proteins.Comment: 6 pages, 7 figure

Odorant binding proteins : a biotechnological tool for odour control

The application of an odorant binding protein for odour control and fragrance delayed release from a textile surface was first explored in this work. Pig OBP-1 gene was cloned and expressed in Escherichia coli , and the purified protein was biochemically characterized. The IC50 values(concentrations of competitor that caused a decay of fluorescence to half-maximal intensity) were determined for four distinct fragrances, namely, citronellol, benzyl benzoate,citronellyl valerate and ethyl valerate. The results showed a strong binding of citronellyl valerate,citronellol and benzyl benzoate to the recombinant protein, while ethyl valerate displayed weaker binding. Cationized cotton substrates were coated with porcine odorant binding protein and tested for their capacity to retain citronellol and to mask the smell of cigarette smoke. The immobilized protein delayed the release of citronellol when compared to the untreated cotton. According to a blind evaluation of 30 assessors, the smell of cigarette smoke, trapped onto the fabrics’ surface, was successfully attenuated by porcine odorant binding protein (more than 60 % identified the weakest smell intensity after protein exposure compared to β-cyclodextrin-treated and untreated cotton fabrics). This work demonstrated that porcine odorant binding protein can be an efficient solution to prevent and/orremove unpleasant odours trapped on the large surface of textiles. Its intrinsic properties make odorant binding proteins excellent candidates for controlled release systems which constitute a new application for this class of proteins.This work was co-funded by the European Social Fund through the management authority POPH and FCT. The authors Carla Silva and Teresa Matama would like to acknowledge their post-doctoral fellowships: SFRH/BPD/46515/2008 and SFRH/BPD/47555/2008, respectively

Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors

Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors, with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer thrombin-binding aptamer (TBA), has been widely explored concerning both its structure, which was resolved with different techniques, and its function, especially about the possibility of using it as the active part of biosensors. This paper proposes a microscopic model of the electrical properties of TBA and the aptamer-thrombin complex, combining information from both structure and function. The novelty consists in describing both the aptamer alone and the complex as an impedance network, thus going deeper inside the issues addressed in an emerging electronics branch known as proteotronics. The theoretical results are compared and validated with Electrochemical Impedance Spectroscopy measurements reported in the literature. Finally, the model suggests resistance measurements as a novel tool for testing aptamer-target affinity.Comment: 5 figures, 1 tabl

Odorant binding proteins fromHermetia illucens: potential sensing elements for detecting volatile aldehydes involved in early stages of organic decomposition

Organic decomposition processes, involving the breakdown of complex molecules such as carbohydrates, proteins and fats, release small chemicals known as volatile organic compounds (VOCs), smelly even at very low concentrations, but not all readily detectable by vertebrates. Many of these compounds are instead detected by insects, mostly by saprophytic species, for which long-range orientation towards organic decomposition matter is crucial. In the present work the detection of aldehydes, as an important measure of lipid oxidation, has been possible exploiting the molecular machinery underlying odour recognition inHermetia illucens(Diptera: Stratiomyidae). This voracious scavenger insect is of interest due to its outstanding capacity in bioconversion of organic waste, colonizing very diverse environments due to the ability of sensing a wide range of chemical compounds that influence the choice of substrates for ovideposition. A variety of soluble odorant binding proteins (OBPs) that may function as carriers of hydrophobic molecules from the air-water interface in the antenna of the insect to the receptors were identified, characterised and expressed. An OBP-based nanobiosensor prototype was realized using selected OBPs as sensing layers for the development of an array of quartz crystal microbalances (QCMs) for vapour phase detection of selected compounds at room temperature. QCMs coated with four recombinantH. illucensOBPs (HillOBPs) were exposed to a wide range of VOCs indicative of organic decomposition, showing a high sensitivity for the detection of three chemical compounds belonging to the class of aldehydes and one short-chain fatty acid. The possibility of using biomolecules capable of binding small ligands as reversible gas sensors has been confirmed, greatly expanding the state-of the-art in gas sensing technology

Biotechnological applications of mammalian odorant-binding proteins

The olfactory system of mammals allows the detection and discrimination of thousands of odors from the environment. In mammals, odorant-binding proteins (OBPs) are considered responsible to carry odorant molecules across the aqueous nasal mucus to the olfactory receptors (ORs). The three-dimensional structure of these proteins presents eight antiparallel ?-sheets and a short ?-helical segment close to the C terminus, typical of the lipocalins family. The great ability of OBPs to bind differentiated ligand molecules has driven the research to understand the mechanisms underlying the OBP function in nature and the development of advanced biotechnological applications. This review describes the role of mammalian OBPs in the olfactory perception, highlighting the influence of several key parameters (amino acids, temperature, ionic strength, and pH) in the formation of the OBP/ligand complex. The information from the literature regarding OBP structure, affinity, the strength of binding, and stability inspiring the development of several applications herein detailed.This study was financially supported by the Portuguese Foundation for Science and Technology (Fundac¸~ao para a Ciencia e a Tecnologia) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation [NORTE-01-0145-FEDER-000004] funded by European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. F.G. thanks Fundac~ao para a Ci^ encia e a Tecnologia for their funding [SFRH/BD/114684/2016].info:eu-repo/semantics/publishedVersio

Perspectives of Nano-Materials and Nanobiosensors in Food Safety and Agriculture

Nanobiosensor is one type of biosensor made up with usage of nanomaterials i.e., nanoparticles and nanostructures. Because of the nanomaterials’ unique properties such as good conductivity, and physicochemical, electrochemical, optical, magnetic and mechanical properties, Nanobiosensors are highly reliable and more sensitive in biosensing approaches over conventional sensors which is having various limitation in detection. Quantum dots, nanotubes, nanowires, magnetic and other nanoparticles enhance sensitivity and lower limit of detection by amplifying signals and providing novel signal transduction mechanisms enable detection of a very low level of food contaminants, pesticides, foodborne pathogens, toxins and plant metabolites. Nanobiosensors are having a lot of scope in sustainable agriculture because of its detecting ability i.e., sensing changes occurred in molecular level. So it can be utilized to find out the variations or modification of plant metabolities, volatiles, gas exchange, hormonal and ion concentration etc. which are the indicators of various harsh environmental stresses (abiotic), biotic and physiological stress. Identification of the stress in the starting stage itself will help us to avoid intensive plant damage and prevent yield losses created by the stress. Nanosensors can be used in smart farming, in which all the environmental factors related to plant growth like temperature, water, pH, humidity, nutritional factor etc. are measured and precaution taken to control the factors which reduce the crop production with the help of IOT platform, thereby enhance the productivity. In this review, discussed about nanobiosensors for detection of food contaminants and various application and its potential in agriculture

Hierarchy and assortativity as new tools for affinity investigation: the case of the TBA aptamer-ligand complex

Aptamers are single stranded DNA, RNA or peptide sequences having the ability to bind a variety of specific targets (proteins, molecules as well as ions). Therefore, aptamer production and selection for therapeutic and diagnostic applications is very challenging. Usually they are in vitro generated, but, recently, computational approaches have been developed for the in silico selection, with a higher affinity for the specific target. Anyway, the mechanism of aptamer-ligand formation is not completely clear, and not obvious to predict. This paper aims to develop a computational model able to describe aptamer-ligand affinity performance by using the topological structure of the corresponding graphs, assessed by means of numerical tools such as the conventional degree distribution, but also the rank-degree distribution (hierarchy) and the node assortativity. Calculations are applied to the thrombin binding aptamer (TBA), and the TBA-thrombin complex, produced in the presence of Na+ or K+. The topological analysis reveals different affinity performances between the macromolecules in the presence of the two cations, as expected by previous investigations in literature. These results nominate the graph topological analysis as a novel theoretical tool for testing affinity. Otherwise, starting from the graphs, an electrical network can be obtained by using the specific electrical properties of amino acids and nucleobases. Therefore, a further analysis concerns with the electrical response, which reveals that the resistance sensitively depends on the presence of sodium or potassium thus posing resistance as a crucial physical parameter for testing affinity.Comment: 12 pages, 5 figure