Ultrafast Microfluidic Immunoassays Towards Real-time Intervention of Cytokine Storms

Biomarker-guided precision medicine holds great promise to provide personalized therapy with a good understanding of the molecular or cellular data of an individual patient. However, implementing this approach in critical care uniquely faces enormous challenges as it requires obtaining “real-time” data with high sensitivity, reliability, and multiplex capacity near the patient’s bedside in the quickly evolving illness. Current immunodiagnostic platforms generally compromise assay sensitivity and specificity for speed or face significantly increased complexity and cost for highly multiplexed detection with low sample volume. This thesis introduces two novel ultrafast immunoassay platforms: one is a machine learning-based digital molecular counting assay, and the other is a label-free nano-plasmonic sensor integrated with an electrokinetic mixer. Both of them incorporate microfluidic approaches to pave the way for near-real-time interventions of cytokine storms. In the first part of the thesis, we present an innovative concept and the theoretical study that enables ultrafast measurement of multiple protein biomarkers (<1 min assay incubation) with comparable sensitivity to the gold standard ELISA method. The approach, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA) incorporates the single-molecular counting of proteins at the early, pre-equilibrium state to achieve the combination of high speed and sensitivity. We experimentally demonstrated the assay’s application in near-real-time monitoring of patients receiving chimeric antigen receptor (CAR) T-cell therapy and for longitudinal serum cytokine measurements in a mouse sepsis model. In the second part, we report the further development of a machine learning-based PEdELISA microarray data analysis approach with a significantly extended multiplex capacity using the spatial-spectral microfluidic encoding technique. This unique approach, together with a convolutional neural network-based image analysis algorithm, remarkably reduced errors faced by the highly multiplexed digital immunoassay at low analyte concentrations. As a result, we demonstrated the longitudinal data collection of 14 serum cytokines in human patients receiving CAR-T cell therapy at concentrations < 10pg/mL with a sample volume < 10 µL and 5-min assay incubation. In the third part, we demonstrate the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a compact fluorescence optical scanner for the potential near-bedside readout. The automated system has achieved high interassay precision (~10% CV) with high sensitivity (<0.4pg/mL). Our data revealed large subject-to-subject variability in patient responses to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays. Lastly, an AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biosensing device was presented for rapid analysis of cytokine IL-1β among sepsis patients. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The technologies developed have enabled new capabilities with broad application to advance precision medicine of life-threatening acute illnesses in critical care, which potentially will allow the clinical team to make individualized treatment decisions based on a set of time-resolved biomarker signatures.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163129/1/yujing_1.pd

Microfluidics for Biosensing

There are 12 papers published with 8 research articles, 3 review articles and 1 perspective. The topics cover: Biomedical microfluidics Lab-on-a-chip Miniaturized systems for chemistry and life science (MicroTAS) Biosensor development and characteristics Imaging and other detection technologies Imaging and signal processing Point-of-care testing microdevices Food and water quality testing and control We hope this collection could promote the development of microfluidics and point-of-care testing (POCT) devices for biosensing

Micro- and Nanofluidics for Bionanoparticle Analysis

Bionanoparticles such as microorganisms and exosomes are recoganized as important targets for clinical applications, food safety, and environmental monitoring. Other nanoscale biological particles, includeing liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicines. The recent deveopment of microfluidic and nanofluidic technologies has enabled the separation and anslysis of these species in a lab-on-a-chip platform, while there are still many challenges to address before these analytical tools can be adopted in practice. For example, the complex matrices within which these species reside in create a high background for their detection. Their small dimension and often low concentration demand creative strategies to amplify the sensing signal and enhance the detection speed. This Special Issue aims to recruit recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and detection of bionanoparticles

Lab-on-a-chip Thermoelectric and Solid-phase Immunodetection of Biochemical Analytes and Extracellular Vesicles: Experimental and Computational Analysis

Microfluidics is the technology of controlling and manipulating fluids at the microscale. Microfluidic platforms provide precise fluidic control coupled with low sample volume and an increase in the speed of biochemical reactions. Lab-on-a-chip platforms are used for detection and quantification of biochemical analytes, capture, and characterization of various proteins, sensitive analysis of cytokines, and isolation and detection of extracellular vesicles (EVs). This study focuses on the development of microfluidic and solid-phase capture pin platforms for the detection of cytokines, extracellular vesicles, and cell co-culture. The fabrication processes of the devices, experimental workflows, numerical analysis to identify optimal design parameters, and reproducibility studies have been discussed. Layer-by-layer assembly of polyelectrolytes has been developed to functionalize glass and stainless-steel substrates with biotin for the immobilization of streptavidinconjugated antibodies for selective capture of cytokines or EVs. Microstructure characterization techniques (SEM, EDX, and fluorescence microscopy) have been implemented to assess the efficiency of substrate functionalization. A detailed overview of current methods for purification and analysis of EVs is discussed as well. Additionally, the dissertation demonstrates the feasibility of a calorimetric microfluidic immunosensor with an integrated antimony-bismuth (Sb/Bi) thermopile sensor for the detection of cytokines with picomolar sensitivity. The developed platform can be used for the universal detection of both exothermic or endothermic reactions. A three-dimensional numerical model was developed to define the critical design parameters that enhance the sensitivity of the platform. Mathematical analyses identified the optimal combinations of substrate material and dimensions that will maximize the heat transfer to the sensor. Lab-on-a-chip cell co-culture platform with integrated pneumatic valve was designed, numerically characterized, and fabricated. This device enables the reversible separation of two cell culture chambers and serves as a tool for the effective analysis of cell-to-cell communication. Intercellular communication is mediated by extracellular vesicles. A protocol for the functionalization of stainless-steel probe with exosomespecific CD63 antibody was developed. The efficiency of the layer-by-layer deposition of polyelectrolytes and the effectiveness of biotin and streptavidin covalent boding were characterized using fluorescent and scanning electron microscopy

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications

DEVELOPMENT OF MICROFLUIDIC PLATFORMS AS A TOOL FOR HIGH-THROUGHPUT BIOMARKER SCREENING

Droplet microfluidic platforms are in the early stages of revolutionizing high throughput and combinatorial sample screening for bioanalytical applications. However, many droplet platforms are incapable of addressing the needs of numerous applications, which require high degrees of multiplexing, as well as high-throughput analysis of multiple samples. Examples of applications include single nucleotide polymorphism (SNP) analysis for crop improvement and genotyping for the identification of genes associated with common diseases. My PhD thesis focused on developing microfluidic devices to extend their capabilities to meet the needs of a wide array of applications

Multi-Functional System for Biomedical Application Using AC Electrokinetics

Manipulation of fluids in a small volume is often a challenge in the field of Microfluidics. While many research groups have addressed this issue with robust methodologies, manipulating fluids remains a scope of study due to the ever-changing technology (Processing Tools) and increase in the demand for “Lab-On-a-Chip” devices in biological applications. This thesis peruses the flow pattern of the orthogonal electrode pattern and circular electrode providing, examples of the flow patterns and the process micromixing. Characteristics of a multifunctional system were demonstrated using orthogonal electrode and circular electrode patterned device. Conductivity of the fluids were chosen such they reflect perfect biological conditions to determine the working conditions of the proposed devices under different AC voltage and frequency levels. Experimental results were then compared with simulated results which were obtained using COMSOL simulation software

Fully integrated microsystem for bacterial genotyping

Methods for bacterial detection and identification has garnered renewed interest in recent years due to the infections they may cause and the antimicrobial resistances they can develop, the potential for bioterrorism threats and possible contamination of food/water supplies. Therefore, the rapid, specific and accurate detection of pathogens is crucial for the prevention of pathogen-related disease outbreaks and facilitating disease management as well as the containment of suspected contaminated food and/or water supplies. In this dissertation an integrated modular-based microfluidic system composed of a fluidic cartridge and a control instrument has been developed for bacterial pathogen detection. The integrated system can directly carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and can detect sequence variations in selected genes to allow for the identification of the bacterial species and even its strain. The unique aspect of this fluidic cartridge is its modular format with a task-specific module interconnected to a fluidic motherboard to permit the selection of a material appropriate for the given processing step(s). In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the fluidic cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and assembled into a small footprint instrument. The fluidic cartridge was capable of performing cell lysis, solidphase extraction of genomic DNA from the whole cell lysate, continuous flow PCR amplification of specific gene fragments, continuous flow ligase detection reaction to discriminate sequence variations and universal DNA array readout, which consisted of DNA probes patterned onto a planar polymer waveguide for evanescent excitation. The performance of the fluidic system was demonstrated through its successful application to the genetic detection of bacterial pathogens, such as Escherichia coli O157:H7, Salmonella, methicillin-resistant Staphylococcus aureus and multi-drug resistant Mycobacterium tuberculosis, which are major threats for global heath. The modular system, which could successfully identify several strains of bacteria in \u3c40 min with minimal human intervention and also perform strain identification, represents a significant contribution to pathogen detection

Magnetic components and microfluidics optimization on a Lab-on-a-chip platform

Tese de mestrado integrado, Engenharia Biomédica e Biofísica (Sinais e Imagens Médicas), Universidade de Lisboa, Faculdade de Ciências, 2017Desde 1934, quando Moldovan criou o primeiro instrumento que poderia ser descrito como um citómetro de fluxo, este equipamento tornou-se um importante componente em várias especialidades dentro do laboratório clínico para o diagnóstico, prognóstico e monitorização de um número incontável de doenças. Esta tecnologia biofísica suspende entidades biológicas num fluxo de fluido, sinalizando-as usando reconhecimento biomolecular, para depois as detetar através de um aparelho de detecção eletrónica. Com o crescimento das técnicas de fabricação de semicondutores e microfluídos, foram e continuam a ser feitas muitas tentativas de criar citómetros de fluxo do tipo Lab-on-a-Chip (LOC), o que certamente irá afastar os equipamentos usados hoje me dia nos laboratórios por equipamentos usados in situ de custo e tamanho reduzidos, portáteis e sem necessidade de pessoal especializado. Após uma revisão bibliográfica das técnicas e princípios de funcionamento dos equipamentos já existentes foi possível perceber que a utilização de partículas magnéticas (PM) pode ter várias vantagens quando comparadas com o uso convencional de deteção por fluorescência, removendo assim a necessidade de integrar e alinhar componentes ópticos, permitindo uma medição direta e a construção de um citómetro de fluxo LOC com preparação, separação e deteção de amostras totalmente magnético. No INESC-MN foi feito um protótipo que permite a deteção de um tipo de PMs em tempo real a velocidades da ordem de cm/s usando sensores magnetoresistivos integrados em canais microfluídicos mas a primeira demonstração desta técnica para aplicações de citómetro foi realizada através da detecção de células Kg1-a marcadas com PMs de 50 nm que passaram, através de um canal microfluídico, sobre 3 sensores magnetoresistivos demonstrando que, para amostras de elevada concentração, pode ter a mesma eficiência que um hemocitómetro, mas com menor erro. Tendo como ambição um dispositivo LOC capaz de contar várias entidades biológicas na mesma amostra, um módulo de contagem com vários canais paralelos é necessário. Nesse sentido, foi projetado um novo chip com 4 colunas separadas por 3 mm, cada uma com 7 sensores do tipo válvula de spin (SV) com uma área de deteção de 100x4 μm2 distanciados 150 μm uns dos outros. Os sensores são abordados individualmente por uma linha de corrente de alumínio de 300 nm e passivados com 300 nm de nitreto de silicio. Alinhados com as colunas de sensores, 4 canais de polydimethylsiloxane (PDMS) com uma secção de 20 μm de altura e 100 μm de largura foram irreversivelmente colados ao chip por ultravioleta-ozono (UVO) criando o canal onde a amostra irá fluir. Para que as PMs sinalizem a sua passagem é necessário colocá-las sob um campo magnético forte o suficiente para induzir a sua magnetização e para que, consequentemente, as PMs emanem um campo marginal significativo. Aproveitando a insensibilidade das SVs às componentes perpendiculares ao seu plano (xy), aplica-se um campo magnético nesse sentido (z) para magnetizar as partículas. As PMs ao passarem sobre o sensor geraram um sinal bipolar devido ao campo marginal criado pela sua magnetização perpendicular. Como é apresentado na simulação do sinal, a amplitude do mesmo depende apenas da altura da partícula em relação ao sensor e da magnetização das mesmas, idealmente, uma saturação da magnetização das partículas e o máximo de proximidade aos sensores geraria a maior amplitude possível. O campo magnético perpendicular foi criado usando um magnete de neodímio posicionado sob a placa de circuito impresso (PCB), onde o chip do citómetro é colado e as ligações entre o chip e a PCB soldadas por ultrassons com fio de alumínio. Na abordagem usada em Loureiro et al., 2011, um magnete de 20 mm x 10 mm x 1 mm foi simplesmente colado sob a PCB, mas devido aos campos magnéticos serem sempre fechados as componentes x e y criam desvios nas curvas de transferência dos sensores deixando apenas 1 ou 2 sensores de uma coluna do chip operacionais. Numa abordagem seguinte foi usado um magnete 20 mm x 20 mm x 3 mm distanciado 2 cm abaixo da PCB, isto tornou as curvas de transferência dos sensores adequadas para medição, mas fez com que a componente z do campo magnético não fosse grande o suficiente para que as PMs emanassem um campo magnético suficientemente forte. Percebendo as falhas de cada uma das configurações anteriores, foram feitas simulações do campo magnético que iria influenciar o chip originado por magnetes de vários tamanhos a várias distancias para perceber qual conseguiria fornecer uma maior área em que as componentes x e y fossem menores que 10 Oe e em que a componente z fosse de pelo menos 1 kOe. Através das simulações foi concluído que o magnete de 20 mm x 10 mm x 1 mm o mais próximo possível do chip seria a melhor solução, mas que um alinhamento preciso seria necessário. Para esse fim, foi fabricado numa fresadora um sistema de alinhamento em PMMA. Para que o alinhamento fosse o correto foram feitos 4 furos de alinhamento no sistema de PMMA e na PCB e para reduzir a distancia ao máximo foi feita uma bolsa na PCB da mesma área que o chip deixando a distancia do magnete aos sensores de 1 mm (0.3 mm de PCB + 0.7 mm de substrato de silício). Com isto, o alinhamento em x foi conseguido, mas para alinhar em y foi criado um trilho no sistema de PMMA onde o magnete pudesse deslizar, controlando-o pela rotação de um parafuso com passo de 0.5 mm. Para colocar o magnete na posição ideal, foi medida consecutivamente a curva de transferência do 4º sensor de uma das colunas, num campo magnético de -141 Oe a 141 Oe, até que este tivesse um campo de acoplamento efectivo (Hf) de aproximadamente 0 Oe, o que significa que a curva de transferência estaria perfeitamente centrada em zero e criaria um sinal bipolar perfeito. Após o alinhamento e posicionamento do magnete, todos os sensores foram caracterizados e, nesses resultados, podemos ver perfeitamente o efeito das componentes x e y do magnete. Com o lado longo do magnete paralelo ao lado longo das SVs e alinhado de forma que o Hf fosse o mais próximo de 0 Oe no 4º sensor de uma coluna, percebemos que a componente x (lado longo) do campo magnético criado pelo magnete tem efeitos na sensibilidade dos sensores fazendo com que esta caia à medida que nos afastamos do centro do magnete. Enquanto que a componente y tem efeitos sobre o Hf dos sensores tornando-o mais positivo à medida que medimos a 3ª, 2ª e 1ª linha de sensores e tornando-o mais negativo quando medimos a 5ª, 6ª e 7ª linha. São também apresentadas simulações dos canais microfluídicos para perceber como a velocidade das partículas afeta o sinal e qual a velocidade máxima permitida para que placa de aquisição eletrónica seja capaz de o detetar. Com estas conclusões, um novo chip foi desenhado e fabricado. Neste novo chip a distância entre as colunas de SVs foi reduzida para apenas 1 mm, o que obrigou também à alteração dos canais microfluídicos, ao tamanho do chip e da estrutura de PDMS. Também são apresentadas simulações que mostram que se um segundo magnete, alinhado com o primeiro, for colocado sobre os canais microfluídicos poderá melhorar a magnetização e a homogeneidade do campo, o que permitirá que os 4 canais tenham a mesma sensibilidade e um desvio padrão de Hf menor. Todos os antecedentes teóricos, os métodos de microfabricação e técnicas de caracterização usados são apresentados e descritos.The diagnosis, prognosis and monitoring of diseases serves for the only purpose of preserving and improving life. Being this the greatest objective of the human kind, since ever that efforts have been made to better our ways to do that. One of those, a very important component in several specialties within the clinical laboratory is the flow cytometer, a biophysical technology which uses biomolecular recognition to sort and count biological entities by suspending them in a stream of fluid and detecting them through an electronic detection apparatus. The improvement of the semiconductor and microfluidic fabrication techniques have created the chance to bring the expensive, specialized and bulky equipment out of the laboratories and generate new machines able of having the same efficiency but with smaller price, size, allowing portability and removing the need for specialized personnel. This is the concept behind the next generation of in pointof- care apparatus, the La-on-a-Chip (LOC). At INESC-MN it is understood the potential that magnetic particles (MP) have in a LOC flow cytometer and as such a real-time detection of single magnetic particles magnetoresistive based cytometer was prototyped. Demonstration of this technique for cytometer applications was accomplished by indicating that for high concentration samples it can have the same efficiency as the hemocytometer method but with lesser error. This thesis has as objective the optimization of the magnetic and microfluidic components of a LOC to allow the parallelization of measurements and enabling the real-time measurement of different particles at the same time. For this purpose, a bibliographic review of the theoretical backgrounds, of the fabrication and characterization techniques, of the different detecting principles and of the already existing magnetoresistive counting modules was made to get a deeper understanding of the optimization possibilities. The present work describes the above-mentioned platform for dynamic detection of magnetic labels with a magnetoresistive based flow cytometer, where a permanent magnet is used to magnetize the labels enabling them to trigger the sensor. Several simulations of the magnetic fields created by the permanent magnet and the microfluidic channels were done and analyzed in order to characterize the MPs signal, understand which would be the best positioning of these components and which fluid velocities would be in the range of the electronic read-out capabilities. This study led to the fabrication of a micromachined polymethylmethacrylate (PMMA) alignment system to correctly position the permanent magnet under the cytometer’s chip. This made the control over the magnet’s positioning more sensible and thus reducing the influence of its unwanted magnetic components on the chip. The approximation of the magnet to the chip enhanced the signal by optimizing the MPs magnetization and consequently the signal amplitude, the precise alignment corrected the sensors response by improving its sensitivity and removing them from saturation states. Through this new setup all the sensors in the chip became operational. Finally, using the several techniques of microfabrication also describe in this thesis, a new chip was designed and fabricated to improve even more the sensors sensitivity and consequently augment the number of the cytometer’s counting channels