Computer vision approach for the determination of microbial concentration and growth kinetics using a low cost sensor system

The measurement of microbial contamination is of primary importance in different fields, from environmental monitoring to food safety and clinical analysis. Today, almost all microbiology laboratories make microbial concentration measurements using the standard Plate Count Technique (PCT), a manual method that must be performed by trained personnel. Since manual PCT analysis can result in eye fatigue and errors, in particular when hundreds of samples are processed every day, automatic colony counters have been built and are commercially available. While quick and reliable, these instruments are generally expensive, thus, portable colony counters based on smartphones have been developed and are of low cost but also not accurate as the commercial benchtop instruments. In this paper, a novel computer vision sensor system is presented that can measure the microbial concentration of a sample under test and also estimate the microbial growth kinetics by monitoring the colonies grown on a Petri dish at regular time intervals. The proposed method has been in-house validated by performing PCT analysis in parallel under the same conditions and using these results as a reference. All the measurements have been carried out in a laboratory using benchtop instruments, however, such a system can also be realized as an embedded sensor system to be deployed for microbial analysis outside a laboratory environment

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

Using functionalized nanoparticles as sensors for rapid monitoring of drinking water quality

In this thesis, filed-deployable nano sensors for onsite detection of nitrite and microorganisms were developed. The colorimetric nitrite ion sensor was designed utilizing 4-aminothiophenol (4-ATP) modified gold nanorods (GNR). In the presence of nitrite ions, the deamination reaction was induced by heating the 4-ATP modified GNR in ethanol solution, resulting in the reduction of the GNR surface charges, which led to aggregation of GNRs and a colorimetric response that was quantitatively correlated to the concentration of nitrite ions. This simple assay was rapid (less than 10 minutes) and highly sensitive (\u3c 1 ppm of nitrite), it can be used for rapid monitoring of drinking water quality. Anisotropic nanoparticles (i.e., silver nanocubes, gold nanorods) based SERS molecular probe were also designed and fabricated for the rapid and specific detection of bacterial targets in a test-in-a-tube platform utilizing a novel dual-recognition mechanism. The probes were synthesized by covalently attaching Raman tags and bacteria-specific antibodies to the surface of silver nanocubes. Specific binding between the probes and bacterial targets ensured surface enhanced Raman spectroscopic (SERS) signatures of the targets to be observed alongside with the SERS signals of the Raman tags. The assessment through the dual signals (superimposed target and tag Raman signatures) established a specific recognition of the targets in a single step, no washing/separation steps were needed to separate target-bound probes from unbound ones, because unbound probes only yield tag signatures, and could easily be distinguished from the target-bound ones. The dual-recognition protocol implemented with a portable Raman spectrometer would become an easy-to-use, field-deployable spectroscopic sensor for onsite detection of pathogenic microorganisms

Electrical Impedance Spectroscopy (EIS) characterization of saline solutions with a low-cost portable measurement system

Electrical Impedance Spectroscopy (EIS), a powerful technique used for wide range of applications, is usually carried out by means of benchtop instrumentation (LCR meters and ìmpedance analyzers), not suited for in-the-field measurements performed outside a laboratory.In this paper a new portable electronic system for EIS on liquid and semi-liquid media is presented that is capable to produce an electrical fingerprint of the sample under investigation. The proposed system was used for the characterization of four different saline solutions (NaCl, Na2CO3, K2HPO4 and CuSO4). A multi-frequency approach, based on the measurement of maximum value of the impedance imaginary component and its corresponding frequency, was tested for the first time to discriminate different saline solutions. The results show that the proposed method is capable to discriminate the different solutions and to measure the concentration (R2 = 0.9965) independently of the type of saline solution. Keywords: Impedance Spectroscopy, Measurement, Frequency, Sensor, Portable syste

Electroanalytical Sensors and Devices for Multiplexed Detection of Foodborne Pathogen Microorganisms

The detection and identification of pathogen microorganisms still rely on conventional culturing techniques, which are not suitable for on-site monitoring. Therefore, a great research challenge in this field is focused on the need to develop rapid, reliable, specific, and sensitive methods to detect these bacteria at low cost. Moreover, the growing interest in biochip development for large scale screening analysis implies improved miniaturization, reduction of analysis time and cost, and multi-analyte detection, which has nowadays become a crucial challenge. This paper reviews multiplexed foodborne pathogen microorganisms detection methods based on electrochemical sensors incorporating microarrays and other platforms. These devices usually involve antibody-antigen and DNA hybridization specific interactions, although other approaches such as the monitoring of oxygen consumption are also considered

A multiplex self-referencing detection of pathogens using surface enhanced raman scattering nanoprobes with a nano-DEP microfluidic concentrator

In this dissertation, I successfully developed the multiplex self-referencing SERS pathogen (E.coli O157: H7) detection biosensor platform which offers high sensitivity (10^1 CFU/mL) and strain level discrimination by measuring the superimposed SERS signatures with multiple characteristic peaks. To harvest the effective Raman molecular probes, I developed methods to fabricate anisotropic metallic nanoparticles to serve as SERS enhancers, and more importantly, I developed surface modification methodology to add functionality to the SERS enhancers so that they can bind specifically to their pathogen targets for highly accurate and sensitive detection. Gold nanorods (GNRs) and gold/silver nanocages are successfully fabricated with high particle yield. Three highly effective linker molecules (4-Aminothiophenol (4-ATP), 3-Amino-1,2,4-triazole-5-thiol (ATT), and 3-Mercaptopropionic acid (3-MPA)) are identified and designed to conjugate on gold nanostructures, and then different monoclonal antibody molecules are designed to bond to the different linkers through diazo-histine binding (4-ATP and ATT) and EDC/NHS bonding (3-MPA-antibody). In addition, this platform demonstrated excellent separation and concentration capacities by using DEP microfluidic devices and further improves the sensitivity to 10^0 CFU/mL. The integration of microfluidic devices with SERS detection has yielded simple and miniaturized instrumentation that is suitable for the detection and characterization of small volume of chemical and biological analytes with high sensitivity and specificity. For data analysis, various preprocessing methods are used for spectral background removal, baseline correction, smoothing, and normalization. Principle Component Analysis (PCA) is applied to reduce the variable dimensions. A Support Vector Machine (SVM) discriminant analysis model based on statistical multivariate model is being developed for superimposed spectra classification. The validation of spectral classification model (target binding VS no target binding) is evaluated by the accuracy percentage, which is above 95%

Smartphone as a Portable Detector, Analytical Device, or Instrument Interface

The Encyclopedia Britannia defines a smartphone as a mobile telephone with a display screen, at the same time serves as a pocket watch, calendar, addresses book and calculator and uses its own operating system (OS). A smartphone is considered as a mobile telephone integrated to a handheld computer. As the market matured, solid-state computer memory and integrated circuits became less expensive over the following decade, smartphone became more computer-like, and more more-advanced services, and became ubiquitous with the introduction of mobile phone networks. The communication takes place for sending and receiving photographs, music, video clips, e-mails and more. The growing capabilities of handheld devices and transmission protocols have enabled a growing number of applications. The integration of camera, access Wi-Fi, payments, augmented reality or the global position system (GPS) are features that have been used for science because the users of smartphone have risen all over the world. This chapter deals with the importance of one of the most common communication channels, the smartphone and how it impregnates in the science. The technological characteristics of this device make it a useful tool in social sciences, medicine, chemistry, detections of contaminants, pesticides, drugs or others, like so detection of signals or image

The EcoChip : a wireless multi-sensor platform for comprehensive environmental monitoring

This paper presents the EcoChip, a new system based on state-of-the-art electro-chemical impedance (EIS) technologies allowing the growth of single strain organisms isolated from northern habitats. This portable system is a complete and autonomous wireless platform designed to monitor and cultivate microorganisms directly sampled from their natural environment, particularly from harsh northern environments. Using 96-well plates, the EcoChip can be used in the field for realtime monitoring of bacterial growth. Manufactured with highquality electronic components, this new EIS monitoring system is designed to function at a low excitation voltage signal to avoid damaging the cultured cells. The high-precision calibration network leads to high-precision results, even in the most limiting contexts. Luminosity, humidity and temperature can also be monitored with the addition of appropriate sensors. Access to robust data storage systems and power supplies is an obvious limitation for northern research. That is why the EcoChip is equipped with a flash memory that can store data over long periods of time. To resolve the power issue, a low-power microcontroller and a power management unit control and supply all electronic building blocks. Data stored in the EcoChip’s flash memory can be transmitted through a transceiver whenever a receiver is located within the functional transmission range. In this paper, we present the measured performance of the system, along with results from laboratory tests in-vitro and from two field tests. The EcoChip has been utilized to collect bio-environemental data in the field from the northern soils and ecosystems of Kuujjuarapik and Puvirnituq, during two expeditions, in 2017 and 2018, respectively. We show that the EcoChip can effectively carry out EIS analyses over an excitation frequency ranging from 750 Hz to 10 kHz with an accuracy of 2.35%. The overall power consumption of the system was 140.4 mW in normal operating mode and 81 µW in sleep mode. The proper development of the isolated bacteria was confirmed through DNA sequencing, indicating that bacteria thrive in the EcoChip’s culture wells while the growing conditions are successfully gathered and stored

Integrated Electrochemical Biosensors for Detection of Waterborne Pathogens in Low-Resource Settings

More than 783 million people worldwide are currently without access to clean and safe water. Approximately 1 in 5 cases of mortality due to waterborne diseases involve children, and over 1.5 million cases of waterborne disease occur every year. In the developing world, this makes waterborne diseases the second highest cause of mortality. Such cases of waterborne disease are thought to be caused by poor sanitation, water infrastructure, public knowledge, and lack of suitable water monitoring systems. Conventional laboratory-based techniques are inadequate for effective on-site water quality monitoring purposes. This is due to their need for excessive equipment, operational complexity, lack of affordability, and long sample collection to data analysis times. In this review, we discuss the conventional techniques used in modern-day water quality testing. We discuss the future challenges of water quality testing in the developing world and how conventional techniques fall short of these challenges. Finally, we discuss the development of electrochemical biosensors and current research on the integration of these devices with microfluidic components to develop truly integrated, portable, simple to use and cost-effective devices for use by local environmental agencies, NGOs, and local communities in low-resource settings