Nanofluidic Actuation of Molecules and Colloids by Controlling Multiphasic Intermembrane Transports

Sangjin Seo Ulsan National Institute of Science and Technology 2024 국내박사

Surface-dominated physicochemical phenomena assume a pivotal role in mediating the migration of fluids and suspended or dissolved solutes within nanopores, exhibiting exquisite sensitivity to external stimuli. These nanofluidic phenomena propel not only the fluids themselves but also transport dissolved solutes. Activating nanopores provides precise control of dissolved solutes by modulating the nanopore’s characteristics, such as pore dimensions and surface properties. The solutes encompass nanometer-scale colloids and highly diffusive molecules in diverse configurations, spanning biomolecular to engineered materials. Nanofluidic actuation — encompassing gating, accumulation, and pumping mechanisms — offers both fundamental and advanced methods of microfluidic manipulation. Gating provides foundational controllability over fluidic transport through on-off switching. Accumulation proves a tool to manage low-concentration solutes for targeted ion detection and sample pre-concentration. Meanwhile, the ion pump is utilized to heighten concentration differences of diffusive molecules. These nanofluidic actuations offer the robustness and versatility of fluidic devices, enriching their unique functions and applications. Microfluidics predominantly favors liquid-only systems due to their viscosity-driven benefits. However, the extension of microfluidics with the gas phase overcomes its traditional liquid-centric precepts, embracing a more expansive understanding of "fluid." Incorporating gases into microfluidics involves trade-offs between durable operation and effective transport. However, phase separation in membrane-integrated microfluidic devices enables various functions in both durable and effective manners. These functions enrich versatilities across multiple fields, including biology, chemistry, and physics. The primary objective of this research is to explore an innovative approach for manipulating small molecules and colloids within a microfluidic chip using liquid-gas interphase transport mechanisms. The improvement of precision and labor-efficiency could lead to significant advancements. Spontaneous processes such as pervaporation and diffusion are designed in a guided manner to achieve improvements without relying on external energy sources. Therefore, the primary focus is on designing and fabricating a membrane-integrated microfluidic device. Nanostructures are further integrated to obtain the capability of molecular and colloidal manipulation. The devices are utilized for various purposes depending on the target. First, pervaporation is utilized to control the transport of small molecules along nanoslits. The local and independent switching of humidity conditions near nanoslits facilitates the concentration, separation, and actuation of small molecules. Pervaporation-induced flow of solvent and diffusion of small molecules along nanoslits are analyzed via fluorescent signals and theoretical modeling simultaneously to investigate critical parameters. These parameters are concluded to provide insightful control of small molecules. Second, a pervaporation-assisted method for fabricating a particle assembly membrane (PAM) is developed. The concentrating property of pervaporation-induced flow is utilized to in-situ assemble sub-micron to nano-sized particles in microchannels. The assembled particles containing nanopore networks serve as nanoporous membranes. Forced assembly due to liquid flow enables the adoption of various types of particles in terms of size, surface functionality, and wettability. Furthermore, the heterogeneous structure in parallel and serial configurations offers possibilities for numerous applications. Third, gas dissolution is employed to modulate the functions of ionic diodes. Asymmetrically charged heterogeneous PAMs serve as ionic diodes. A control channel is placed in the middle of the ionic diode to supply gas molecules through diffusion via a gas-permeable film. Gases and gas- dissolved solutions are introduced to construct a concentration gradient in a switchable and programmable manner. The diodes exhibit different responses to bias when gas is supplied from the control channel, resulting in the modulation of rectification ratios. Several demonstrations are also conducted, including chemical reactions in an accumulated state and ion signal amplification. In summary, the methods of fabrication and control serve as the basis for designing liquid-gas interphase transport in an on-chip manner. Furthermore, this research accelerates molecular, colloidal, and ionic manipulation in microfluidic devices, thereby enriching micro/nanofluidics and various fields. Spontaneous working mechanisms, especially, enhance the key value of microfluidics, offering low cost and portability. A wide range of target molecules enables the development of multi-functional devices for practical applications, such as cell culture, chemical reactions/sensing, and colloidal delivery.

Fully integrated smart microfluidic platform for in situ exosome analysis and classification : 통합형 스마트 미세 유체 플랫폼을 이용한 현장 엑소좀 분석 및 분류

김준수 경북대학교 대학원 2025 국내박사

With the growing need for early-stage disease diagnosis, research has been in- creasingly focused on analyzing the physical properties of biomarkers. Although conventional optical methods offer high sensitivities, they face miniaturization challenges due to their reliance on external light sources. Microfluidic resistive pulse sensing provides a simpler alternative for detecting and analyzing micropar- ticles in various fields such as environmental, chemical, biomedical, and disease diagnostics. This study introduces a microfluidic platform designed to analyze and identify the physical properties of cancer-derived exosomes. Fluid injection within the microfluidic chip is fully automated, utilizing a capillary- and vacuum-chamber- assisted passive-driven technology. To ensure precise measurements, particles are hydrodynamically focused without the need for a sheath, and a reference gate is used to minimize noise during resistive pulse detection. Using this microfluidic ap- proach, the proposed microfluidic chip accurately measures the size, concentration and zeta potential of exosomes derived from MCF-7, MDA-MB-231, and MCF- 10A cell lines with high sensitivity. Additionally, deep learning algorithms are ap- plied to classify and identify the exosomes based on the collected data with accu- racy of 96.6%. The microfluidic platform offers high sensitivity in the analysis and classification of exosome physical properties, making it suitable for potential uses in in vitro clinical applications. Keywords: Microfluidics, Exosome Analysis, Resistive Pulse Sensing, Ma- chine Learning 질병 조기 진단의 필요성이 증가함에 따라 바이오마커의 물리적 특성을 분석하는 연구가 활발히 이루어지고 있다. 기존의 광학적 방법들은 높은감도를 제공하지만 외부 광원에 의존한다는 점에서 소형화에 어려움을 겪는다. 저항성 펄스 센싱은 환경, 화학, 생물의학, 질병 진단 등 다양한 분야에서 미세입자를 감지하고 분석하는 간단한 대안을 제공한다. 본 연구에서는 암유래 엑소좀의 물리적 특성을 분석하고 식별하기 위해 설계된 미세유체플랫폼을 소개한다. 미세유체 칩 내의 유체 주입은 모세관 현상 및 진공챔버를 이용해 자동화가 가능하다. 정밀한 측정을 위해, 입자의 위치는 유체 역학적으로 제어되며, 저항 펄스 감지 중 잡음을 최소화하기 위해 참조게이트가 사용된다. 이 미세유체 접근 방식을 사용하여 제안된 미세유체 칩은MCF-7, MDA-MB-231, 및 MCF-10A 세포에서 유래된 엑소좀의 크기, 농도 및 제타 전위를 높은 감도로 정확하게 측정한다. 또한, 수집된 데이터를 기반으로 엑소좀을 분류하고 식별하기 위해 딥러닝 알고리즘을 적용하여 96.6%의 정확도를 달성한다. 이 미세유체 플랫폼은 엑소좀의 물리적 특성을 분석하고 분류하는 데 높은 감도를 제공하여 체외 임상 응용에 적합하다.

Microfluidic Technologies to Advance Antibody and Bacteriophage Discovery

Keepseagle, Kayla E Harvard University ProQuest Dissertations & Theses 2024 해외박사(DDOD)

This dissertation introduces a pioneering approach to antibody discovery that harnesses the power of advanced microfluidic technology. Our innovative methodology revolves around the isolation of singular antibody-producing B-cells, a feat accomplished through the encapsulation of individual B-cells and an oligo-dT gel within microdroplets. This cutting-edge technique enables the targeted capture of a single type of antibody, thereby streamlining the discovery process. Leveraging microfluidic systems, we have meticulously designed a microenvironment conducive to encapsulating a solitary B-cell alongside the oligo-dT gel. The microdroplets function as miniature reaction vessels, providing an isolated setting for antibody synthesis and capture. This microscale strategy not only enhances efficiency but also minimizes sample requirements. Furthermore, we have employed state-of-the-art DNA sequencing techniques to meticulously analyze and characterize the antibody library resulting from our microfluidic-assisted capture. The comprehensive DNA sequencing results have furnished invaluable insights into the diversity and specificity of the isolated antibodies. The pivotal findings of this research underscore the potential of our microfluidic-assisted method for expeditious and precise antibody discovery. By capturing antibodies at the single B-cell level, we have ushered in a new era of possibilities for generating targeted and highly specific antibodies. This work serves as a significant contribution to the advancement of antibody engineering and lays the groundwork for the development of therapeutic agents boasting enhanced efficacy and precision.This research explores an innovative system for phage therapy leveraging microfluidics to revolutionize the precision and efficacy of bacterial infection treatment. Through a startup experiment, we designed a microfluidic platform where bacterial cells were encapsulated in microdroplets, each serving as an independent microenvironment for targeted phage therapy. Confocal microscopy was employed to visualize and analyze the dynamic interactions within these microdroplets. Bacterial cells were introduced into microdroplets, creating a controlled environment where some droplets contained bacteriophages, while others did not. This experimental design facilitated real-time observation and quantification of the impact of phage therapy at the microscale. The use of microfluidics allowed for the encapsulation of individual bacterial cells, providing a unique perspective on the dynamics of phage-bacterium interactions. The success of this experimental setup demonstrates the potential of microfluidics in advancing phage therapy precision. The targeted nature of phage therapy, observed and quantified at the microscale, opens avenues for developing tailored and responsive treatments for bacterial infections. These findings underscore the transformative potential of microfluidic-enabled phage therapy in addressing antibiotic resistance challenges and enhancing treatment precision. The promising outcomes of this startup experiment pave the way for further exploration and refinement of microfluidic-assisted phage therapy. Future research endeavors may focus on scaling up the system, optimizing parameters for broader applications, and exploring the clinical translatability of this precision medicine approach.

Microfluidic macroemulsion stabilization through In situ interfacial coacervation of associative nanoplatelets and polyelectrolytes

Kim, Hajeong Sungkyunkwan University 2022 국내석사

This study generated a facile and useful methodology for stabilizing macroemulsions through the in situ coacervation of associative silica nanoplatelets (ASNPs) and poly (acrylic acid) (PAA) at the oil–water (O/W) interface. To produce monodisperse macroemulsion droplets in the length scale near 1000 μm, the inner fluid containing partially positively charged ASNPs and the outer fluid dissolving negatively charged PAA were coflowed through a capillary-based microfluidic channel. The generation of a bilayered coacervate at the O/W interface was confirmed by direct observation of confocal laser scanning microscopy. Dynamic interfacial tension and interfacial rheology measurements revealed that the migration of ASNPs and PAA from each phase to the interface led to the formation of a complex bilayered thin membrane with an enhanced interfacial modulus, thereby structurally stabilizing these large drops. In addition, we demonstrated that adjusting the surface properties of ASNPs by coupling a fluorochemical enabled the production of monodisperse fluorocarbon-in-oil-in-water double macroemulsions. These results highlighted the applicability of our microfluidics-based interfacial coacervation technology in the development of complex fluid products with visual differentiation and drug encapsulation. 본 연구에서는 유–수 계면에서 자기회합 실리카 나노판형입자 (ASNP)와 폴리 아크릭액씨드 (PAA)의 계면 코아세르베이션을 통해 거대액적을 안정화하는 쉽고 유용한 시스템을 제안한다. 1000 μm에 가까운 균일한 거대액적을 생성하기 위해 부분적으로 양전하를 띤 ASNP를 포함하는 분산상과 음전하를 띤 PAA가 분산되어 있는 연속상을 함께 미세 유체 채널에 함께 통과시켰다. 유–수 계면에서 이중층 코아세르베이트의 형성은 공초점레이저 주사현미경의 관찰에 의해 확인되었다. 동적 계면장력 분석을 통해, ASNP와 PAA가 각 상에서 계면으로 이동에 의한 얇은 이중층의 복합 계면막을 형성된다는 것을 확인하였다. 이어진 계면 유변학 연구에서는 이중층 계면막의 강한 물성이 마크로에멀젼을 성공적으로 안정화 시킬 수 있다는 것을 규명하였다. 또한, 우리는 불소실란의 커플링 반응을 통해 ASNP의 표면 특성을 개질함으로써 F/O/W 이중 마크로에멀젼의 제조 역시 가능하다는 것을 입증하였다. 이러한 결과는 우리의 미세유체기술 기반 계면 코아세르베이션 기술이 화장품 산업에서 복합 유체 제품의 개발이나 제약 산업에서의 약물 캡슐화에 적용될 수 있을을 보여준다.

Leveraging Microfluidics and Electrokinetics to Improve Sample Preparation and Biomarker Detection

Yost, Jarad Wilker Texas A&M University ProQuest Dissertations & Thes 2023 해외박사(DDOD)

With the advent of global pandemics, the world has developed a demonstrable need for rapid and low-cost testing platforms for diagnostics. Microfluidic diagnostics can meet this demand due to the versatility and scalability of microfluidic devices. However, two components of microfluidic diagnostics, sample preparation and biomarker detection, require further innovation for effective use within microfluidics. In this work, improvements to microfluidic sample preparation and biomarker detection are presented. To improve microfluidic sample preparation, the leading electrolyte (LE) in free-flow isotachophoresis (FFITP) was replaced with a conductive wall, limiting system complexity and analyte-electrolyte interactions. This new system, called free-flow teichophoresis (FFTPE), was used to concentrate protein, separate multiple proteins, and concentrate nucleic acids. To improve microfluidic biomarker detection, a novel heating method called Electrokinetic Nucleic Acid Amplification (E-NAAMP) was developed. E-NAAMP replaces traditional boundary-driven heating techniques found in microfluidic nucleic acid amplification (NAA) by applying electric current directly to the reaction. E-NAAMP was used to drive NAA using both Loop-Mediated Isothermal Amplification (LAMP) and the Polymerase Chain Reaction (PCR). Finally, improvements were made to E-NAAMP by incorporating it into a novel paper microfluidic platform called Microfluidic Pressure-in-Paper (μPiP). Paper E-NAAMP was used to amplify nucleic acids with LAMP. It was also demonstrated that paper passivation via a carrier protein is necessary for paper E-NAAMP success. With these improvements, we envision an all-in-one chip where FFTPE is used to remove NAA inhibitors from biological samples and E-NAAMP is subsequently used to amplify these inhibitor-free nucleic acid samples.

(A) study on electrowetting devices for efficient digital microfluidics

장종현 Graduate School, Korea University 2010 국내박사

Digital microfluidics (DMF) is an alternative technology for lab-on-a-chip systems, in which the microfluidic functions of discrete fluid droplets could be performed without microchannel networks or mechanical moving parts. Electrowetting is an attractive method to manipulate liquid droplets in DMF because it allows all the microfluidic functions of dispensing, transporting, splitting, merging, and mixing the fluid droplets with the advantages such as the absence of heat generation, rapid switching response, and low power consumption. The objectives of this study are to review electrowetting components and configurations for DMF and to improve efficiency of the electrowetting devices for low-voltage operation, simple fabrication, or high electrowetting force generation. An electrowetting device consists of four compulsory components of electrodes, a conducting liquid droplet surrounded by an insulating medium, a dielectric insulator, and a hydrophobic layer. Design criteria for successful droplet transport by electrowetting were described. The electrode width and spaces of permanent hydrophobic regions between two control electrodes should be considered with the droplet size. The required voltage can be reduced by decreasing the liquid-vapor interfacial tension using surfactant-assisted droplets or surrounding medium immiscible with the liquid droplet. However, the choice of surfactant or surrounding medium should be considered from the targeted application. The properties of dielectric materials have a remarkable impact on electrowetting performance. When selecting dielectric material, its electrical, mechanical, thermal, chemical properties should be considered. Atomic layer deposition (ALD) is one of the best methods to deposit a pinhole-free and thin conformal dielectric layer. The ALD Al2O3 was employed as the dielectric layer for low voltage electrowetting. After the control electrode array of 1 mm × 1 mm squares with 50 μm spaces was patterned on a glass substrate, 127 nm thick Al2O3 (εr = 10.4) film was grown by ALD system. Then, a 20 μm wide reference electrode line was patterned on the Al2O3 film and 30 nm thick Teflon AF was spin-coated. The minimum voltage to move 2 μL water droplet could be as low as 3 V, which is the lowest threshold voltage reported so far in electrowetting researches. This result opens a possibility of manipulating droplets without any surfactant or oil treatment at only a few volts using ALD Al2O3 as the electrowetting dielectric. The hydrophobic surface is essential for enlarging the contact angle variation with high inherent contact angle and reducing contact angle hysteresis, which makes the droplet manipulation more efficient in electrowetting devices. The thinner hydrophobic layer is coated on the dielectric layer, the better electrowetting efficiency is when using a thin dielectric layer. An effective and efficient condition for Teflon AF spin-coating was obtained experimentally, and the resulting minimum acceptable Teflon AF concentration and thickness were 0.2 wt% and 9 nm respectively for large inherent contact angle of about 116° and small contact angle hysteresis of 4-6°. Electrowetting configurations for DMF are divided into roughly two-plate and single-plate configurations. The two-plate electrowetting device is suitable for a wide range of droplet operations such as dispensing, transporting, splitting, and merging. In the ground-type two-plate configuration, the electrowetting force is generated only on the bottom plate. When the top plate is replaced by the same as the bottom plate, the electrowetting force for droplet transport can be generated on both plates. However, the applied voltage is divided into two dielectric layers. Therefore, the force in the unground-type two-plate electrowetting decreases. Single-plate configurations also can be divided into ground-type and unground-type. As well as the two-plate configurations, the ground-type is more efficient in electrowetting performance than the unground-type, but it requires an additional fabrication step for patterning the top reference electrode on the dielectric layer. The proposed configuration for single-plate electrowetting devices allows more simplified fabrication process without any decrease of the electrowetting performance. The control electrode array and the reference electrode were patterned simultaneously on the same plane. Then, polyimide as the dielectric layer was patterned for opening the reference electrode and then Teflon AF was coated. The droplet movement of diluted methylene blue by electrowetting was successfully demonstrated. A novel two-plate electrowetting configuration was proposed to enhance the electrowetting efficiency from the configurational point of view. This twin-plate configuration, a combination of two same ground-type single-plate devices, allows applying the voltage across each of the dielectric layers on both plates without any voltage division, while keeping the droplet grounded with the reference electrodes. Therefore, the total electrowetting force can increase 2-fold compared to a typical two-plate configuration theoretically. For making the twin-plate electrowetting device, two same fabricated ground-type single-plate devices were attached with a gap between them after aligning their control electrode arrays to face each other with one of them upside down. This twin-plate electrowetting device shows significantly faster droplet velocity than that of the two-plate device, and hence the required voltage for generating a desired electrowetting force can be reduced to below 80%. Although the contact angle changes when an electrowetting force is applied to the droplet on the control electrode array, the droplet cannot move onto the activated control electrode with small applied voltage below a certain threshold because of the adhesive friction. In order to transport the droplet successfully, sufficient contact angle difference between the advancing side and the receding side should be generated by an applied voltage over the threshold voltage. The dependence of the threshold voltage for droplet transport on the contact angle hysteresis was described theoretically and experimentally for three different ground-type electrowetting configurations. Analyzing the contact angles at the electrowetting threshold voltage theoretically, it was found that the threshold voltage in either the single-plate or twin-plate configuration equals to the hysteresis voltage and it becomes higher than that of the two-plate configuration by factor of square root of 2. The fabricated devices using 2.5 μm thick polyimide (εr = 3.3) as the dielectric layer and thin Teflon AF as the hydrophobic layer (θ0 = 116° and α = 4-6°) showed the threshold voltages of 28, 40, and 28 V for droplet transport initiation, and the threshold voltages of 35, 50, and 35 V for stable droplet transport in the single-plate, two-plate, and twin-plate devices, respectively. All the results are in very good agreement with theoretical expectations.

(A) study on stretchable microfluidic-integrated biosensor patch for wearable point-of-care testing

Bae, Chanwool Sungkyunkwan University 2022 국내박사

As the demand for personalized preventive medicine through smart healthcare has exploded, the development of wearable POCT system which can continuously monitor personal physiological information such as respiration, heart rate, skin temperature, body motion, and bio-markers in biofluid, and diagnose the personal health status in real-time, is highly required. Among the various components of wearable POCT system, wearable biosensor recently has received tremendous attention as they can continuously and directly detect biomarkers in biofluid, which is closely related to health status. However, there are still several problems such as insufficient stretchability, poor detection accuracy, dependence on detection environment, and biofluid mixing effect, to be solved for implementation of perfect non-invasive and real-time continuous detection. The stretchability of wearable biosensor is very important not only for preventing noise signal generated by body movements, but also for conformal attachment of wearable biosensor with body to continuously collect biofluid. Therefore, to impart stretchability to non-stretchable materials which are essential for the implementation of high-performance wearable biosensor, the biosensors are directly fabricated on the mogul-patterned substrate which has three-dimensional micro-pattern with continuously connected bumps and valleys in three directions, effectively absorbing the stress generated during physical deformation. In addition, the nanomaterials which have high surface to volume ratio and great chemical stability, is used as catalyst instead of bioreceptor to enhance the detection accuracy and stability against to interfering molecules and detection environment respectively. As a result, stretchable electrochemical and fuel cell-based glucose biosensors with catalytic nanomaterials, which is fabricated on mogul-patterned substrate show high sensitivity to glucose, high selectivity against to interfering molecules, great multi-directional stretchability, and low dependance on detection environments such as pH and temperature. Furthermore, to prevent the biofluid mixing effect due to the continuous extraction from the body, stretchable cotton materials which is inexpensive, durable, biocompatible, and has great fluid absorption capability even under its stretched condition, based microfluidic devices are developed. The fabricated stretchable cotton materials based microfluidic devices show excellent fluid handling characteristics with constant and stable efficiency even under the various physical deformations. Then, fully stretchable microfluidic-integrated biosensor patches are developed by assembling the stretchable biosensors with stretchable microfluidic device. The fabricated fully stretchable microfluidic-integrated biosensor patches achieve high detection accuracy and reliability through precisely collect and handle the sweat, effectively preventing biofluid mixing effect. Finally, daily sweat glucose level monitoring is perfectly conducted through real-time continuous on-body detection with a fully stretchable biosensor patches attached onto the skin. In conclusion, the results of this study will open up new horizons of wearable biosensor platforms for personalized preventive medicine by addressing the currently existing limitations in real-time continuous on-body detection. 스마트 헬스케어를 통한 맞춤형 예방의학에 대한 수요가 폭발적으로 증가함에 따라, 생체액의 호흡, 심박수, 피부온도, 신체 움직임, 체액내의 바이오 마커 등 생리정보를 지속적으로 모니터링하고, 개인의 건강상태를 실시간으로 진단할 수 있는 웨어러블 POCT 시스템의 개발이 절실히 요구되고 있습니다. 다양한 웨어러블 POCT 시스템 요소들 중, 웨어러블 바이오센서는 건강 상태와 아주 밀접한 관련이 있는 체액내의 바이오 마커를 지속적이고 직접적으로 감지할 수 있기 때문에 최근 엄청난 주목을 받고 있으나, 완벽한 실시간 연속 검출을 구현하기 위해서는 불충분한 신축성, 낮은 검출 정확도, 검출 환경에 대한 의존성 및 체액 혼합현상 같은 해결해야 할 몇 가지 문제가 여전히 남아있습니다. 웨어러블 바이오 센서의 신축성은 신체 움직임으로 인한 노이즈 신호발생을 방지할 뿐만 아니라 체액을 지속적으로 수집하기 위한 피부와의 등각 부착에도 매우 중요합니다. 따라서 비신축성 특성을 가진 소재에 신축성을 부여하기 위해, 바이오센서는 세방향으로 언덕과 골이 연속적으로 연결되어 있는 입체 마이크로 패턴을 가져, 물리적 변형에서 발생하는 응력을 효과적으로 흡수할 수 있는 모굴 기판에 제작되었습니다. 또한, 높은 표면적 대 부피비와 뛰어난 화학적 안정성을 갖는 나노 물질을 간섭 분자에 대한 검출 정확도와 검출 환경에 대한 검출 안정성을 향상시키기 위해 바이오 리셉터 대신 촉매로 사용하였습니다. 결과적으로, 모굴 패턴 기판 상에 제작된 나노촉매물질을 사용한 신축성 포도당 바이오센서들은 포도당에 대한 높은 감도, 간섭 분자에 대한 높은 선택성, 뛰어난 다방향 신축성 및 pH나 온도와 같은 검출 환경에 대한 낮은 의존성을 보여주었습니다. 다음으로, 연속적인 추출로 인한 체액 혼합을 방지하기 위해, 저렴하고 내구성과 생체 적합성이 뛰어나며 다양한 신축조건에서도 안정적으로 유체를 흡수하는 능력을 가진 신축성 면 소재 기반의 미세유체소자들이 개발하였습니다. 제작된 면 소재를 기반 미세유체소자들은 다양한 물리적변형에도 일정하고 안정적인 효율의 유체 처리 특성을 보여주었습니다. 마지막으로, 완전히 신축성이 있는 미세유체소자 통합 바이오센서 패치는 신축성 바이오센서를 신축성 미세유체소자와 집적하여 제작되었으며, 땀을 정밀하게 수집 및 처리하여 체액의 혼합 효과를 효과적으로 방지를 통해 높은 검출 정확도 및 신뢰도를 이뤄냈습니다. 결론적으로, 본 연구의 결과들은 현재 존재하는 실시간 연속 신체감지의 한계점들을 해결함으로써 개인화 된 예방 의학을 위한 웨어러블 바이오 센서플랫폼의 새로운 지평을 열어줄 것입니다.

Multiplex Analysis System Integrated with Air Sampler and DC Impedance-based Microfluidic Cytometer

장우혁 서울대학교 대학원 2020 국내박사

This dissertation is a study on the development of multiplex analytical platform equipped with air sampling device and microfluidic cytometer. There are the increasing demands for chemical analysis in everyday life and various field. In particular, multiplex assay has become important for point-of-care testing and biosurveillance because it enables a rapid, low-cost, and reliable quantification. In this work, I will demonstrate the use of a microfluidic cytometry platform based on DC impedance to conduct multiplex immunoassays and combine with air sampling. First, DC impedance-based flow cytometry using virus-tethered gold microspheres was utilized for multiplex immunoassays. For common use of bead-based multiplex immunoassays, enhanced sensitivity, effective prevention of non-specific adsorption and miniaturization of the detection device are required. In this work, we have conducted multiplex immunoassay applications employing virus-tethered gold microspheres and a DC impedance-based microfluidic cytometer. The merits of virus-tethered gold microspheres are excellent prevention of non-specific adsorption in biological fluid and signal enhancement arising from the large quantity of antibody loading on each virus. Using these merits, a microfluidic chip-based flow cytometer detected DC impedance and fluorescence signals of virus-tethered gold microspheres for detection and quantification of biomarkers. This platform successfully realized multiplex immunoassays involving four biomarkers: cardiac troponin I (cTnI), prostate specific antigen (PSA), creatine kinase MB (CK-MB), and myoglobin in intact human sera, enhancing sensitivity by up to 5.7-fold compared to the gold microspheres without virus. Constructive integration between filamentous virus-tethered gold microspheres and use of a microfluidic cytometer suggests a promising strategy for multiplex immunoassay development based on bead-based immunoassays. Second, DC impedance-based microfluidic cytometer was utilized for bioaerosol detection using wet-cyclone air sampler. We present a bioaerosol detection system consisting of a DC impedance based microfluidic cytometer and wet cyclone air sampler. Microbeads and bacteria E. coli are uniformly dispersed into an air chamber and collected by wet-cyclone air sampler. The collected liquid sample is transferred to the microfluidic cytometer using syringe pump and valve. Then, the microfluidic cytometer measured size and concentration of collected particles. The microfluidic cytometer was validated by analyzing the diameters of microbeads ranging from 0.96 to 2.95 µm and concentrations of microbeads ranging from 1 × 103 mL-1 to 1 × 107 mL-1. Simultaneous measurement of DC impedance and fluorescence using microfluidic cytometer implemented detection of collected particles and E. coli, demonstrating capability to measure concentration of particle similar to commercial instrument FACS. From these results, the detection strategy of airborne particle using microfluidic cytometer is established for biosurveillance. The developed components show that the microfluidic-based cytometry platform is capable of multiplexing and measuring aerosols of bacteria by combination with air sampler. The developed components based on DC impedance microfluidic cytometry will be the cornerstone of the integrated total analytical platform with other microfluidic system.

Development of a microfluidics-based tooth-pulp flow phantom for validation of doppler ultrasound devices

김도현 Graduate School, Yonsei University 2016 국내박사

I. Introduction Doppler ultrasound is generally used in medical diagnostics for measuring blood flow in a wide range of blood vessels. Recently, Doppler ultrasound has been used for measuring pulpal blood flow (PBF). However, the reliability of this method has not been sufficiently addressed. Flow phantoms have been used to evaluate the velocity estimation using Doppler ultrasound devices. However, most of these phantoms are designed to simulate relatively large blood vessels in milli or centimeter scale, which passes through the matrix material that mimics soft tissue. Therefore, they are not applicable as substitutes for dental hard tissues and pulp. Microfluidics is known as the science of fluid mechanics manipulated at micro or nanometer scales. It has emerged as an important tool in several research fields for microanalytical purposes. There have been a few studies using microfluidics for simulating tissue-vascular network. However, the use of microfluidics for evaluating Doppler ultrasound devices, as well as dental hard tissue and pulp has not been reported. In this study, we present a microfluidics-based flow phantom developed as a blood flow model of dental hard tissue and pulp for use in experiments involving Doppler ultrasound technique. By using the flow phantom, the accuracy of a Doppler ultrasound device in making velocity estimations was evaluated. II. Materials and methods A computer-controlled microfluidic system was constructed to generate triangular pulsatile flow profiles. Blood-mimicking fluid was pumped through a 200×200 μm-sized channel in the microfluidic chip. A Doppler ultrasound device with a 20 MHz-transducer was used for the measurement of fluid flow. The peak, mean, and minimal flow velocities obtained from the flow phantom and the Doppler ultrasound device were compared using linear regression analysis and Pearson's correlation coefficient. Bland-Altman analyses were performed to evaluate the differences of the velocities between the phantom and the Doppler ultrasound device. III. Results The microfluidic system was able to generate the flow profiles as intended, and the fluid flow could be easily monitored and controlled by the software program. Using the soft lithography technique, we were able to fabricate a micrometer-sized channel. There were excellent linear correlations between the peak, mean, and minimal flow velocities of the phantom and the measured velocities from the Doppler ultrasound device (r = 0.94, 0.98, and 0.996, respectively, p < 0.001). However, it is observed that the Doppler ultrasound device overestimated the flow velocities by 1.69, 2.00, and 2.23 cm/s, with respect to the peak, mean, and minimal velocities. IV. Conclusions We believe that this phantom provides opportunities for expanding future researches involving Doppler ultrasound, as well as in the field of hemodynamics and physiology of the dental pulp. Although Doppler ultrasound can be an effective diagnostic tool for quantitative measurement of PBF, it is essential to validate and calibrate the system prior to clinical use. I. 서론 도플러 초음파는 의학 영역에서 다양한 혈관의 혈류를 측정하기 위해 사용되고 있다. 최근에 도플러 초음파를 이용하여 치수의 혈류 속도를 측정한 연구 결과들이 보고되고 있으나, 치수 혈류 측정에 있어 도플러 초음파 기기의 사용에 대한 신뢰도는 아직 충분히 검증되지 않았다. 도플러 초음파 기기의 혈류 속도 측정을 평가하기 위해 “flow phantom” 이라는 모형이 사용되고 있다. 하지만 대부분의 팬텀은 연조직 내부를 지나가는 상대적으로 큰 직경의 혈관을 모사하고 있기 때문에, 치아 경조직과 치수를 재현하기에는 부적절하다. 미세유체공학은 마이크로 또는 나도 단위의 유체를 다루는 학문으로, 다양한 연구 분야에 응용되고 있다. 최근 미세유체공학을 이용하여 조직-혈관 네트워크를 재현하기 위한 시도들이 이루어지고 있다. 하지만, 도플러 초음파 기기의 평가 또는 치아 경조직 및 치수의 재현에 사용된 경우는 아직 보고된 바가 없다. 본 연구에서는, 미세유체 시스템을 이용하여 도플러 초음파 기기에 적용가능한 치아-치수 팬텀을 개발하고, 팬텀을 사용하여 도플러 초음파 기기의 혈류 속도 측정 능력을 평가하고자 하였다. II. 재료 빛 방법 미세유체 시스템을 사용하여 다양한 속도의 박동성 유체 흐름을 만들고, 이를 통해 혈류 모사 용액을 미세유체 칩 내부의 200×200 μm 크기의 직선형 유로 내부로 흘려보냈다. 20 MHz-continuous wave transducer가 장착된 도플러 초음파 기기를 사용하여 용액의 유속을 측정하였다. 실제 팬텀에서 흐르는 용액의 최고, 평균, 최저 속도와 도플러 초음파 기기에서 측정된 최고, 평균, 최저 속도를 선형 회귀분석 및 Pearson 상관계수를 통해 분석하였으며, Bland-Altman analysis를 통해 실제 속도와 측정된 속도 간의 차이를 평가하였다. III. 결과 미세유체 시스템을 사용하여 원하는 형태와 속도의 유체 흐름을 생성할 수 있었으며, 컴퓨터 소프트웨어를 통해 실시간 모니터링 및 조절이 가능하였다. Soft lithography 기술을 사용하여, 마이크로미터 크기의 유로 제작이 가능하였다. 도플러 초음파 기기에서 측정된 최고, 평균, 최저 속도는 실제 팬텀 용액의 속도와 높은 상관관계를 나타내었다 (각각 r = 0.94, 0.98, 0.996, p < 0.001). 하지만, 도플러 초음파 기기에서 측정된 속도가 실제 팬텀에서 흘려보낸 속도와 비교하여 과측정되는 경향을 보였다 (최고, 평균, 최저 속도에서 각각 2.23, 2.00, 1.69 cm/s 과측정). IV. 결론 본 연구를 통해 제작된 팬텀은 도플러 초음파를 활용한 연구 및 치수 혈류에 관한 연구에 도움을 줄 수 있을 것으로 기대된다. 도플러 초음파는 치수 혈류 속도를 정량적으로 측정할 수 있는 좋은 진단 도구가 될 수 있지만, 임상에 적용하기 위해서는 기기의 정확도와 유효성에 대한 평가 및 검정이 선행되어야 할 것이다.

Heterogeneous Immunoassay Considerations Towards Point-Of-Care Microfluidics-Based Diagnostics

Hwu, Alexander T University of California, Irvine ProQuest Disserta 2022 해외박사(DDOD)

Numerous non-conventional heterogeneous immunoassay substrates have been developed to improve the capture and detection of biomolecules from a liquid sample, but many remain unutilized for point-of-care, microfluidics-based diagnostics. These non-conventional substrates often exhibit unique behaviors making the universal assay principles less applicable and more challenging to resolve when unexpected results arise during microfluidic integration. This thesis demonstrates one example of integrating a non-conventional immunoassay with microfluidics. A nitrocellulose-based microarray immunoassay is integrated with centrifugal disc (CD) microfluidics. The CD microfluidics was used to generate reciprocating fluid flow over the immunoassay reaction surface to improve the capture of biomolecules during the liquid sample incubation step of the assay. The CD microfluidic flow generation technique was characterized, simulated, and tuned for characteristic flow conditions before testing the immunoassay. Unexpected assay results contradicting theoretical expectations were obtained, requiring additional substrate assay troubleshooting. During the process, the substrate behavior was characterized and interesting revelations specific to its microfluidic integration were discovered. With the newfound knowledge, the assay was retested, and results explained. Ultimately, this study demonstrates the multifaceted complexities when combining two conceptually simple technologies towards point-of-care, microfluidics-based diagnostics.