abstract: This work describes efforts made toward the development of a compact, quantitative fluorescence-based multiplexed detection platform for point-of-care diagnostics. This includes the development of a microfluidic delivery and actuation system for multistep detection assays. Early detection of infectious diseases requires high sensitivity dependent on the precise actuation of fluids. Methods of fluid actuation were explored to allow delayed delivery of fluidic reagents in multistep detection lateral flow assays (LFAs). Certain hydrophobic materials such as wax were successfully implemented in the LFA with the use of precision dispensed valves. Sublimating materials such as naphthalene were also characterized along with the implementation of a heating system for precision printing of the valves. Various techniques of blood fractionation were also investigated and this work demonstrates successful blood fractionation in an LFA. The fluid flow of reagents was also characterized and validated with the use of mathematical models and multiphysics modeling software. Lastly intuitive, user-friendly mobile and desktop applications were developed to interface the underlying Arduino software. The work advances the development of a system which successfully integrates all components of fluid separation and delivery along with highly sensitive detection and a user-friendly interface; the system will ultimately provide clinically significant diagnostics in a of point-of-care device. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018

MSc. Thesis / Introduction: In 2018, the College of Midwives of Ontario expanded the scope of practice for registered midwives to include performing point of care ultrasound (POCUS) to aid their obstetrical clinical assessments. This project evaluated learner sonography knowledge, skill acquisitions, and integration of this technology following an innovative POCUS curriculum developed for midwives to understand the impacts of this training. Methods: Concurrent triangulation with mixed methods was used in this study through surveys and interviews. First, the surveys were used to collect data across four time points including before and after the intervention. Five- and seven-point Likert scale questions were analyzed through descriptive statistics. Open-ended questions were qualitatively analyzed using thematic analyses. Second, the semi-structured interviews were conducted to better understand participants’ attitudes and clinical behaviours. Interviews were coded and analyzed using a combination of Corbin and Strauss as well as Charmaz approaches to grounded theory. Results: The findings demonstrated how there was a positive growth in learner comfort with POCUS and a desire to continue using it during clinical practice. The frequency of POCUS use within antenatal care increased with common applications including fetal presentation and assessment of pregnancy viability. The interviews highlighted five themes: facilitating learning, improving care, refining the role of the midwife, serving community, and maintaining competency. Access to clinical placements and the cost of a device were identified as barriers to promoting POCUS skill sets. Participants who had access to a device and completed the clinical practicum are continuing to use sonography within their clinics to provide more comprehensive client care. Conclusions: This project demonstrates how POCUS training can positively impact midwives particularly with aiding clinical decision making such as those regarding fetal viability and presentation. However, the challenges with obtaining a clinical practicum to ensure competency and the cost and access to a device, are significant barriers that unless they are addressed, may result in minimal integration within community practices. / Thesis / Master of Health Sciences (MSc) / The ability to conduct ultrasounds has recently been included in Ontario midwives’ scope of practice. This project evaluated a newly developed Point of Care Ultrasound (POCUS) curriculum for practicing midwives to understand how midwives can learn and apply POCUS into their clinical work. Learners completed surveys at four time points from pre-course to one-year post-course to share their experiences of knowledge and skill acquisition and applying this training to their clinical work. Five midwives also participated in an interview to share their perspectives about the course more in-depth. It was found that Ontario midwives were very interested to learn POCUS to improve client care. Unfortunately, they struggled finding time and opportunities to refine their sonography skills such as securing a clinical practicum or the funds to purchase a device. However overall, POCUS in midwifery was well received, this technology and its respective education should continue to be supported in Ontario.

Les techniques de biologie moléculaire ont pris au cours des 20 dernières années une place importante dans le diagnostic direct des pathogènes viraux. Notre travail a porté sur la mise en place et le développement d’une plate-forme de biologie moléculaire, au sein du laboratoire de virologie de l’hôpital de la Timone, pour répondre aux demandes et contraintes du diagnostic en milieu hospitalier. L’organisation de cette plate-forme a nécessité plusieurs étapes : la prévention des risques de contamination, l’aliquotage et le stockage des réactifs, l’automatisation des techniques d’extraction des acides nucléiques, la mise au point de témoins positifs synthétiques et de témoins internes et l’optimisation des protocoles de PCR. Cette approche optimisée du diagnostic moléculaire des infections virales a été appliqué notamment à la détection de la grippe pandémique A/H1N1v dans les laboratoires de routine hospitalière et d’urgence « Point Of Care ». La mise en place de cette plate-forme a fait progresser de manière considérable le diagnostic moléculaire du laboratoire. Elle nous permet actuellement de détecter un grand nombre de pathogènes (>80) et de réaliser des tests dans un format à haut débit (≈40 000 tests/an). Au total, cette plateforme est au coeur de la capacité du laboratoire pour réagir de manière rapide aux évènements d'émergence en mettant en place rapidement des procédures diagnostiques standardisées. Ces techniques ont été transférées à de nombreux autres laboratoires de virologie partenaires nationaux et internationaux. Nous envisageons maintenant son utilisation dans une approche syndromique avec notamment, le développement du diagnostic des virus respiratoires. / Molecular biology techniques have taken an important role in the direct diagnosis of viral pathogens over the last 20 years. Our work focused on establishing and developing a platform for molecular diagnosis in the laboratory of Virology (Timone Hospital) to meet the demands and constraints of diagnosis in hospitals. The organization of this platform required several steps: prevention of contamination risks, aliquoting and storage of reagents, automation techniques of nucleic acid extraction, development of synthetic positive controls and internal controls and optimization of PCR protocols. This optimized approach of the molecular diagnosis of viral infections has particularly been applied to the detection of pandemic influenza A/H1N1v in hospital laboratories for routine and emergency "Point Of Care." The implementation of this platform has significantly improved molecular diagnosis in our laboratory. It currently allows us to detect a large number of pathogens (> 80) and perform tests in a high-throughput (≈ 40,000 tests per year). In total, this platform is at the heart of the laboratory capacity to react quickly to emerging events by rapidly implementing standardized procedures. These techniques have been transferred to many other partners’ laboratories nationally and internationally. We are now considering its use in a syndromic approach including the development of the diagnosis of respiratory viruses.

En Afrique, les fièvres récurrentes causées par différentes espèces bactériennes du genre Borrelia sont des infections négligées transmises par les arthropodes et sont responsables de manifestations cliniques variant d’une septicémie mortelle à des formes plus bénignes et d'autres manifestations cliniques, en particulier d'avortement chez les femmes enceintes. Quatre espèces différentes de Borrelia, initialement séparées les unes des autres sur la base de leur répartition géographique et de leur vecteur, sont actuellement cultivées de prélèvements cliniques et de vecteurs : Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. Ces différentes espèces circulent sur le continent africain en parallèle avec au moins six espèces non encore cultivées et détectées dans des vecteurs. Notre travail est une contribution à l’investigation des fièvres récurrentes à Borrelia en Afrique. Dans cette perspective, nous avons mis au point la détection rapide en spectrométrie de masse MALDI-TOF des Borrelia dans les tiques en créant au préalable une base de données Borrelia MALDI-TOF-MS. La base de données de Borrelia et un logiciel de soustraction IHU ont été utilisés pour détecter B. crocidurae dans 20 tiques Ornithodoros sonrai, y compris huit tiques qui ont été testées positives pour B. crocidurae par PCR-séquençage, ce qui ouvre la voie à l'utilisation du MALDI-TOF-MS pour la double identification des vecteurs et des agents pathogènes vectorisés, dont il s’agissait du premier exemple maintenant étendu à d’autres modèles dans notre laboratoire. / In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and other clinical manifestations, particularly abortion in pregnant women. Four different species of Borrelia, initially distinguished one from another on the basis of geography and vector, are currently cultured causative agents in Africa: Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. These different species are circulating in parallel to at least six not-yet cultured species in vectors. Our work consisted in the investigation of recurrent fevers borreliosis in Africa. We have developed rapid detection in MALDI-TOF mass spectrometry of Borrelia in ticks by creating a prior a Borrelia MALDI-TOF-MS database. The Borrelia database and a custom software program that subtracts the uninfected O. sonrai profile were used to detect B. crocidurae in 20 O. sonrai ticks, including eight ticks that tested positive for B. crocidurae by PCR-sequencing; which paves the way for the use of MALDI-TOF-MS for the dual identification of vectors and vectorized pathogens. We have also illustrates a non-specialized circulation of B. crocidurae borreliae within a collection of 35 O. sonrai ticks in West Africa. These ticks were genotyped by 16S rRNA mitochondrial gene sequencing while B. crocidurae was genotyped by Multispacer Sequence Typing (MST). The 35 ticks were grouped into 12 genotypes strong geographic structuring and 35 B. crocidurae into 29 genotypes without strict geographic structure. One O. sonrai genotype carried several B. crocidurae genotypes and one B. crocidurae genotype was found in different O. sonrai genotypes.

Rapid and reliable diagnostics of a disease represents one of the main focuses of today’s academic and industrial research in the development of new sensor prototypes and improvement of existing technologies. With respect to demographic changes and inhomogeneous distribution of the clinical facilities worldwide, especially in rural regions, a new generation of miniaturized biosensors is highly demanded offering an easy deliverability, low costs and sample preparation and simple usage. This work focuses on the integration of nanosized electronic structures for high-specific sensing applications into adequate microfluidic structures for sample delivery and liquid manipulation. Based on the conjunction of these two technologies, two novel sensor platforms were prototyped, both allowing label-free and optics-less electrochemical detection ranging from molecular species to eukaryotic micron-sized human cells.:Table of Figures List of Tables Abbreviations List of Symbols 1 Introduction 1.1 Motivation 1.2 State of the art 1.3 Scope of this thesis 2 Fundamentals 2.1 Sensors at the nanoscale 2.2 Transistors technology 2.2.1 p-n junction 2.2.3 The MOSFET 2.2.4 The ISFET and BioFET 2.3 Impedance measurements for biodetection 2.3.1 Electrical impedance spectroscopy 2.3.2 Electrical impedance cytometry 2.4 Microfluidics 2.4.1 Definition 2.4.2 Droplet-based microfluidics 2.5 Biomarkers for sensing applications 2.5.1 Peripheral blood mononuclear cells (PBMCs) 2.5.2 Physical parameters 3. Material and methods 3.1 General 3.1.1 Materials and chemicals 3.1.2 Surface cleaning 3.2 Lithography 3.2.1 Electron beam lithography 3.2.2 Laser lithography 3.2.3 UV lithography 3.2.4 Soft lithography 3.3 Thermal deposition of metals 3.4 APTES functionalization 3.4.1 Fluorescent labeling of APTES 3.5 Measurement devices 3.5.1 SiNW FET measurements 3.5.2 Electrical Impedance cytometry measurements 3.6 Bacteria and cell cultivation 3.6.1 PBMC purification and treatment 3.6.2 Bacteria cultivation 4. Compact nanosensors probe microdroplets 4.1 Overview 4.2 Fabrication 4.2.1 SiNW FET fabrication 4.2.2 SiNW FET modification for top-gate sensing 4.3 Electrical characterization 4.4 Flow-focusing droplet generation 4.4.1 Flow-focusing geometry 4.4.2 Flow-focusing droplet characterization 4.4.3 Microfluidic integration 4.5 Deionized water droplet sensing 4.6 Phosphate-buffered saline (PBS) droplet sensing 4.6.1 Influence of the droplet’s ionic concentration 4.6.2 Plateau formation in dependence of the droplet’s settling time 4.6.3 Droplet analysis by their ratio 4.6.4 Dependence on pH value 4.6.5 Long time pH sensing experiment 4.6.6 Dependence on ionic concentration 4.7 Tracking of reaction kinetics in droplets 4.7.1 Principle and setup of the glucose oxidase (GOx) enzymatic test 4.7.2 GOx enzymatic assay 4.8 Stable baseline by conductive carrier phase 5. Impedance-based flow cytometer on a chip 5.1 Overview 5.2 Overview of the fabrication of the sensor device 5.3 COMSOL simulation of sensing area 5.3.1 Prototyping of the sensing geometry 5.3.2 Optimization of the sensing geometry 5.3.3 Evaluation of the working potential 5.3.4. Scaling of the sensing area 5.4 Fabrication of the nanoelectronic sensing structure 5.4.1 Nanofabrication and analysis 5.4.2 Evaluation of the proximity effect 5.5 Microcontacting of nanostructured sensing structures 5.6 Electrical characterization of the sensing structure 5.6.1 Characterization in alternating current 5.6.2 Characterization in direct current (DC) 5.7 Scaling effect of nanostructures in static sensing conditions 5.8 Multi-analyte detection on the sensor 5.9 Microfluidic focusing system 5.9.1 1D focusing using FITC-probed deionized water 5.9.2 2D Focusing using fluorescent microparticles 5.10 Microfluidic integration of the two technologies 5.11 Dynamic SiO2 particle detection 5.11.1 Single particle detection 5.11.2 Scatter plot representation 5.11.3 Effect of the sensing area in dynamic particle detection 5.11.4 Dynamic detection of SiO2 particles with different diameters 5.12 Detection of peripheral blood mononuclear cells (PBMCs) 5.12.1 Overview 5.12.2 PBMC classification detected by impedance cytometry 5.12.3 PBMC Long-time detection 5.13 Detection of acute myeloid leukemia by impedance cytometry 5.13.1 Manual analysis of the output response 5.13.2 Learning algorithm for automatic cell classification 5.14 Exploring the detection limit of the device 6. Summary and outlook Scientific output References Acknowledgements / Rasche und zuverlässige biologische Krankheitsdiagnostik repräsentiert eines der Hauptfokusse heutiger akademischer und industrieller Forschung in der Entwicklung neuer Sensor-Prototypen und Verbesserung existierender Technologien. In bezug auf weltweite demographische Änderungen und hohe Distanzen zu Kliniken, besonders in ländlichen Gegenden, werden zusätzliche Anfordungen an neue miniaturisierte Biosensor-Generationen gestellt, wie zum Beispiel ihre Transportfähigkeit, geringe Kosten und Probenpräparation, sowie einfache Handhabung. Diese Dissertation beschäftigt sich mit der Integration nanoskalierter Strukturen zur Detektion chemischer und biologischer Spezies und mikrofluidischen Kanälen zu deren Transport und zur Manipulation der Ströme. Basierend auf der Verbindung dieser beiden Technologien wurden zwei Sensor-Plattformen entwickelt, die eine markierungsfreie und nicht-optische elektrische Detektion von Molekülen bis zu eukaryotischen menschlichen Zellen erlauben.:Table of Figures List of Tables Abbreviations List of Symbols 1 Introduction 1.1 Motivation 1.2 State of the art 1.3 Scope of this thesis 2 Fundamentals 2.1 Sensors at the nanoscale 2.2 Transistors technology 2.2.1 p-n junction 2.2.3 The MOSFET 2.2.4 The ISFET and BioFET 2.3 Impedance measurements for biodetection 2.3.1 Electrical impedance spectroscopy 2.3.2 Electrical impedance cytometry 2.4 Microfluidics 2.4.1 Definition 2.4.2 Droplet-based microfluidics 2.5 Biomarkers for sensing applications 2.5.1 Peripheral blood mononuclear cells (PBMCs) 2.5.2 Physical parameters 3. Material and methods 3.1 General 3.1.1 Materials and chemicals 3.1.2 Surface cleaning 3.2 Lithography 3.2.1 Electron beam lithography 3.2.2 Laser lithography 3.2.3 UV lithography 3.2.4 Soft lithography 3.3 Thermal deposition of metals 3.4 APTES functionalization 3.4.1 Fluorescent labeling of APTES 3.5 Measurement devices 3.5.1 SiNW FET measurements 3.5.2 Electrical Impedance cytometry measurements 3.6 Bacteria and cell cultivation 3.6.1 PBMC purification and treatment 3.6.2 Bacteria cultivation 4. Compact nanosensors probe microdroplets 4.1 Overview 4.2 Fabrication 4.2.1 SiNW FET fabrication 4.2.2 SiNW FET modification for top-gate sensing 4.3 Electrical characterization 4.4 Flow-focusing droplet generation 4.4.1 Flow-focusing geometry 4.4.2 Flow-focusing droplet characterization 4.4.3 Microfluidic integration 4.5 Deionized water droplet sensing 4.6 Phosphate-buffered saline (PBS) droplet sensing 4.6.1 Influence of the droplet’s ionic concentration 4.6.2 Plateau formation in dependence of the droplet’s settling time 4.6.3 Droplet analysis by their ratio 4.6.4 Dependence on pH value 4.6.5 Long time pH sensing experiment 4.6.6 Dependence on ionic concentration 4.7 Tracking of reaction kinetics in droplets 4.7.1 Principle and setup of the glucose oxidase (GOx) enzymatic test 4.7.2 GOx enzymatic assay 4.8 Stable baseline by conductive carrier phase 5. Impedance-based flow cytometer on a chip 5.1 Overview 5.2 Overview of the fabrication of the sensor device 5.3 COMSOL simulation of sensing area 5.3.1 Prototyping of the sensing geometry 5.3.2 Optimization of the sensing geometry 5.3.3 Evaluation of the working potential 5.3.4. Scaling of the sensing area 5.4 Fabrication of the nanoelectronic sensing structure 5.4.1 Nanofabrication and analysis 5.4.2 Evaluation of the proximity effect 5.5 Microcontacting of nanostructured sensing structures 5.6 Electrical characterization of the sensing structure 5.6.1 Characterization in alternating current 5.6.2 Characterization in direct current (DC) 5.7 Scaling effect of nanostructures in static sensing conditions 5.8 Multi-analyte detection on the sensor 5.9 Microfluidic focusing system 5.9.1 1D focusing using FITC-probed deionized water 5.9.2 2D Focusing using fluorescent microparticles 5.10 Microfluidic integration of the two technologies 5.11 Dynamic SiO2 particle detection 5.11.1 Single particle detection 5.11.2 Scatter plot representation 5.11.3 Effect of the sensing area in dynamic particle detection 5.11.4 Dynamic detection of SiO2 particles with different diameters 5.12 Detection of peripheral blood mononuclear cells (PBMCs) 5.12.1 Overview 5.12.2 PBMC classification detected by impedance cytometry 5.12.3 PBMC Long-time detection 5.13 Detection of acute myeloid leukemia by impedance cytometry 5.13.1 Manual analysis of the output response 5.13.2 Learning algorithm for automatic cell classification 5.14 Exploring the detection limit of the device 6. Summary and outlook Scientific output References Acknowledgements

<p>In the year of 2020, an international pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has afflicted tens of millions of people’s life also disrupting global economics. Diagnostic testing is an important part of ensuring public health until a vaccine that has been shown to be safe and effective is made available to the general public. Most tests for detecting COVID-19 utilize quantitative polymerase chain reaction (qPCR) assays, which is a specific and relatively simple quantitative assay that could provide adequate sensitivity for diagnosing early infection. Although powerful, these lab-based molecular assays have a significant lag time, usually several days before receiving results. To satisfy the needs of different purposes (diagnostics, screening, and surveillance), a unified approach is impractical. This thesis presents an alternative testing method supporting the current procedure of point of care (POC) testing and in community testing. This paper-based test overcomes the limitations of current testing methods by utilizing reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and receiving the result on-site by a color change in the presence of the virus within 60 minutes. The test utilizes untreated freshly collected saliva, a less invasive specimen, as the sample and possesses a limit of detection (LoD) of 200 copies of virus per microliter of whole saliva with an analytical sensitivity of 97% and analytical specificity of 100%. The test requires minimal operator training and could be fabricated on a large-scale using roll-to-roll methods. Since the test is based on nucleic acids, the testing platform itself lends to further applications <a>including food safety monitoring, animal diagnostic, etc. simply by changing the specific primers</a>. </p>

Poverty and long distances are two reasons why some people in the third world countries hasdifficulties seeking medical help. A solution to the long distances could be if the medical carewas more mobile and diagnostically tests could be performed on site in villages. A new pointof-care test based on a small blood shows promising results both in run time and mobility.However, the method still needs more advanced equipment for analysis of the resultingmicroarray. This study has investigated the potential to perform the analysis within asmartphone application, performing all steps from image capturing to a diagnostic result. Theproject was approach in two steps, starting with implementation and selection of imageanalysis methods and finishing with implementing those results into an Android application.A final application was not developed, but the results gained from this project indicates that asmartphone processing power is enough to perform heavy image analysis within a sufficientamount of time. It also imply that the resolution in the evaluated images taken with a Nexus 6together with an external macro lens most likely is enough for the whole analysis, but furtherwork must be done to ensure it.

UA Open Access Publishing Fund / Study objectives: Multiple curricula have been designed to teach medical students the basics of ultrasound; however, few focus on critical problem-solving. The objective of this study is to determine whether a theme-based ultrasound teaching session, dedicated to the use of ultrasound in the management of the hypotensive patient, can impact medical students’ ultrasound education and provide critical problem-solving exercises. Methods: This was a cross-sectional study using an innovative approach to train 3rd year medical students during a 1-day ultrasound training session. The students received a 1-hour didactic session on basic ultrasound physics and knobology and were also provided with YouTube hyperlinks, and links to smart phone educational applications, which demonstrated a variety of bedside ultrasound techniques. In small group sessions, students learned how to evaluate patients for pathology associated with hypotension. A knowledge assessment questionnaire was administered at the end of the session and again 3 months later. Student knowledge was also assessed using different clinical scenarios with multiple-choice questions. Results: One hundred and three 3rd year medical students participated in this study. Appropriate type of ultrasound was selected and accurate diagnosis was made in different hypotension clinical scenarios: pulmonary embolism, 81% (95% CI, 73%–89%); abdominal aortic aneurysm, 100%; and pneumothorax, 89% (95% CI, 82%–95%). The average confidence level in performing ultrasound-guided central line placement was 7/10, focused assessment with sonography for trauma was 8/10, inferior vena cava assessment was 8/10, evaluation for abdominal aortic aneurysm was 8/10, assessment for deep vein thrombus was 8/10, and cardiac ultrasound for contractility and overall function was 7/10. Student performance in the knowledge assessment portion of the questionnaire was an average of 74% (SD =11%) at the end of workshop and 74% (SD =12%) 3 months later (P=0.00). Conclusion: At our institution, we successfully integrated ultrasound and critical problemsolving instruction, as part of a 1-day workshop for undergraduate medical education

Background: Goal-directed ultrasound protocols have been developed to facilitate efficiency, throughput, and patient care. Hands-on instruction and training workshops have been shown to positively impact ultrasound training. Objectives: We describe a novel undifferentiated chest pain goal-directed ultrasound algorithm-focused education workshop for the purpose of enhancing emergency medicine resident training in ultrasound milestones competencies. Methods: This was a cross-sectional study performed at an academic medical center. A novel goal-directed ultrasound algorithm was developed and implemented as a model for teaching and learning the sonographic approach to a patient with undifferentiated chest pain. This algorithm was incorporated into all components of the 1-day workshop: asynchronous learning, didactic lecture, case-based learning, and hands-on stations. Performance comparisons were made between postgraduate year (PGY) levels. Results: A total of 38 of the 40 (95%) residents who attended the event participated in the chest pain objective standardized clinical exam, and 26 of the 40 (65%) completed the entire questionnaire. The average number of ultrasounds performed by resident class year at the time of our study was as follows: 19 (standard deviation [SD]=19) PGY-1, 238 (SD=37) PGY-2, and 289 (SD=73) PGY-3. Performance on the knowledge-based questions improved between PGY-1 and PGY-3. The application of the novel algorithm was noted to be more prevalent among the PGY-1 class. Conclusion: The 1-day algorithm-based ultrasound educational workshop was an engaging learning technique at our institution.

In the following work, nanoparticle quantum dot (QD) fluorophores have been exploited to measure biologically relevant analytes via a miniaturized sensor ensemble to provide key diagnostic and prognostic information in a rapid, yet sensitive manner—data essential for effective treatment of many diseases including HIV/AIDS and cancer. At the heart of this “nano-bio-chip” (NBC) sensor is a modular chemical/cellular processing unit consisting of either a polycarbonate membrane filter for cell-based assays, or an agarose bead array for detection of biomarkers in serum or saliva. Two applications of the NBC sensor system are described herein, both exhibiting excellent correlation to reference methods ((R² above 0.94), with analysis times under 30 minutes and sample volumes below 50 [mu]L. First, the NBC sensor was employed for the sequestration and enumeration of T lymphocytes, cells specifically targeted by HIV, from whole blood samples. Several different conjugation methods linking QDs to recognition biomolecules were extensively characterized by biological and optical methods, with a thiol-linked secondary antibody labeling scheme yielding intense, specific signal. Using this technique, the photostability of QDs was exploited, as was the ability to simultaneously visualize different color QDs via a single light pathway, effectively reducing optical requirements by half. Further, T-cell counts were observed well below the 200/[mu]L discriminator between HIV and AIDS and across the common testing region, demonstrating the first reported example of cell counting via QDs in an enclosed, disposable device. Next, multiplexed bead-based detection of cancer protein biomarkers CEA, Her-2/Neu, and CA125 in serum and saliva was examined using a sandwich immunoassay with detecting antibodies covalently bound to QDs. This nano-based signal was amplified 30 times versus molecular fluorophores and cross talk in multiplexed experiments was less than 5%. In addition, molecular-level tuning of recognition elements (size, concentration) and agarose porosity resulted in NBC limits of detection two orders of magnitude lower than ELISA, values competitive with the most sensitive methods yet reported (0.021 ng/mL CEA). Taken together, these efforts serve to establish the valuable role of QDs in miniaturized diagnostic devices with potential for delivering biomedical information rapidly, reliably, and robustly. / text