Surface-based Microfluidic Systems for Enhanced Biomarker Detection

The 21st century has seen a surge in the development of point-of-care (POC) testing systems integrated with microfluidic bioassay devices, for portable, fast, and user-friendly disease diagnostics. These systems detect diagnostic biomarkers from a small quantity of the patient’s blood plasma, by exploiting their innate nature to bind to specific receptor molecules. A microfluidic bioassay device is considered to be of “high efficiency” when low biomarker concentrations (1 pM–1 nM) can be detected within a few minutes. This thesis explores the collective influence of surface chemistry, biomarker transport and biomolecular reactions at the microscale, to propose design principles for the development of rapid, sensitive and user-friendly fluorescence-based POC systems. First, we exploit radio-frequency air plasma to covalently tether receptor proteins within polymethyl methacrylate microfluidic bioassay devices, at high-throughput. Next, these devices are integrated with a palm-sized modular Fluid Handling Device that allows precise mixing, filtration, and delivery of fluids, for subsequent detection of Chlamydia trachomatis specific antibodies, with a limit of detection (LoD) of 7 nM within 15 mins, serving as a “proof-of-concept” POC testing device. Next, biomarker transport-dependent kinetic enhancements in microfluidic bioassay systems are investigated using novel 3D glass devices, where real-time binding events between varying concentrations of fluorescently-labelled receptor and ligand antibodies are analyzed. Combing experimental measurements with scaling analysis, two key control dimensionless parameters are proposed to achieve “rapid” and “sensitive” ligand detection: a local Peclet number P eδ that characterizes the balance between local convection and diffusion-driven transport of ligands; and a kinetic Damkohler number (Dakinetic) that characterizes the balance between the rates of receptor–ligand binding and convection-driven ligand replenishment. We observe that homogeneous ligand binding can be achieved by decreasing the depletion layer thickness (> 105. At Dakinetic > 107 for Dakinetic << 10−2. With prior knowledge of the kinetic constants, these design principles can be applied to various biomolecular systems, paving way to creating highly efficient POC testing systems in the near future.Okinawa Institute of Science and Technology Graduate Universit

Air Plasma-Enhanced Covalent Functionalization of Poly(methyl methacrylate): High-Throughput Protein Immobilization for Miniaturized Bioassays

Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and high-throughput biochemical screening platforms. Owing to its recyclability, and robust mechanical and optical properties, poly(methyl methacrylate) (PMMA) has become the most sought after material for the large-scale fabrication of these platforms. However, the chemical inertness of PMMA entails the use of complex chemical surface treatments for covalent immobilization of proteins. In addition to being hazardous and incompatible for large-scale operations, conventional biofunctionalization strategies pose high risks of compromising the biomolecular conformations, as well as the stability of PMMA. By exploiting radio frequency (RF) air plasma and standard 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry in tandem, we demonstrate a simple yet scalable PMMA functionalization strategy for covalent immobilization (chemisorption) of proteins, such as green fluorescent protein (GFP), while preserving the structural integrities of the proteins and PMMA. The surface density of chemisorbed GFP is shown to be highly dependent on the air plasma energy, initial GFP concentration, and buffer pH, where a maximum GFP surface density of 4 × 10–7 mol/m2 is obtained, when chemisorbed on EDC–NHS-activated PMMA exposed to 27 kJ of air plasma, at pH 7.4. Furthermore, antibody-binding studies validate the preserved biofunctionality of the chemisorbed GFP molecules. Finally, the coupled air plasma and EDC–NHS PMMA biofunctionalization strategy is used to fabricate microfluidic antibody assay devices to detect clinically significant concentrations of Chlamydia trachomatis specific antibodies. By coupling our scalable and tailored air plasma-enhanced PMMA biofunctionalization strategy with microfluidics, we elucidate the potential of fabricating sensitive, reproducible, and sustainable high-throughput protein screening systems, without the need for harsh chemicals and complex instrumentation

Proof‐of‐concept modular fluid handling prototype integrated with microfluidic biochemical assay modules for point‐of‐care testing

Large populations around the world suffer from numerous but treatable health issues, caused by either lifestyle choices or environmental factors. Over the past decades, point-of-care testing kits have been developed to circumvent the reliance on laboratories, by allowing users to perform preliminary health or environmental testing from the privacy of their homes. However, these kits heavily rely on the precision of the user to perform the procedures, leading to increased variability in final assessments. To eliminate user-induced errors, we present an integrated, completely sealed, and disposable point-of-care testing prototype that exploits the benefits of microfluidics and 3D-printing fabrication techniques. The palm-sized modular prototype consists of a manually operated fluid handling device that allows precise mixing, filtration, and delivery of fluids to an on-board microfluidic assay unit for subsequent detection of specific biochemical analytes, with a minimized risk of contamination

Plasma-Assisted Large-Scale Nanoassembly of Metal–Insulator Bioplasmonic Mushrooms

Large-scale plasmonic substrates consisting of metal–insulator nanostructures coated with a biorecognition layer can be exploited for enhanced label-free sensing by utilizing the principle of localized surface plasmon resonance (LSPR). Most often, the uniformity and thickness of the biorecognition layer determine the sensitivity of plasmonic resonances as the inherent LSPR sensitivity of nanomaterials is limited to 10–20 nm from the surface. However, because of time-consuming nanofabrication processes, there is limited work on both the development of large-scale plasmonic materials and the subsequent surface functionalizing with biorecognition layers. In this work, by exploiting properties of reactive ions in an SF₆ plasma environment, we are able to develop a nanoplasmonic substrate containing ∼10⁶/cm² mushroom-like structures on a large-sized silicon dioxide substrate (i.e., 2.5 cm by 7.5 cm). We further investigate the underlying mechanism of the nanoassembly of gold on glass inside the plasma environment, which can be expanded to a variety of metal–insulator systems. By incorporating a novel microcontact printing technique, we deposit a highly uniform biorecognition layer of proteins on the nanoplasmonic substrate. The bioplasmonic assays performed on these substrates achieve a limit of detection of 10^–17 g/mL (∼66 zM) for biomolecules such as antibodies (∼150 kDa). Our simple nanofabrication procedure opens new opportunities in fabricating versatile bioplasmonic materials for a wide range of biomedical and sensing applications

Microcontact printing with aminosilanes: creating biomolecule micro- and nanoarrays for multiplexed microfluidic bioassays

Microfluidic systems integrated with protein and DNA micro- and nanoarrays have been the most sought-after technologies to satisfy the growing demand for high-throughput disease diagnostics. As the sensitivity of these systems relies on the bio-functionalities of the patterned recognition biomolecules, the primary concern has been to develop simple technologies that enable biomolecule immobilization within microfluidic devices whilst preserving bio-functionalities. To address this concern, we introduce a two-step patterning approach to create micro- and nanoarrays of biomolecules within microfluidic devices. First, we introduce a simple aqueous based microcontact printing (μCP) method to pattern arrays of (3-aminopropyl)triethoxysilane (APTES) on glass substrates, with feature sizes ranging from a few hundred microns down to 200 nm (for the first time). Next, these substrates are integrated with microfluidic channels to then covalently couple DNA aptamers and antibodies with the micro- and nanopatterned APTES. As these biomolecules are covalently tethered to the device substrates, the resulting bonds enable them to withstand the high shear stresses originating from the flow in these devices. We further demonstrated the flexibility of this technique, by immobilizing multiple proteins onto these APTES-patterned substrates using liquid-dispensing robots to create multiple microarrays. Next, to validate the functionalities of these microfluidic biomolecule microarrays, we perform (i) aptamer-based sandwich immunoassays to detect human interleukin 6 (IL6); and (ii) antibody-based sandwich immunoassays to detect human c-reactive protein (hCRP) with the limit of detection at 5 nM, a level below the range required for clinical screening. Lastly, the shelf-life potential of these ready-to-use microfluidic microarray devices is validated by effectively functionalizing the patterns with biomolecules up to 3 months post-printing. In summary, with a single printing step, this aminosilane patterning technique enables the creation of functional microfluidic micro- and nano-biomolecule arrays, laying the foundation for high-throughput multiplexed bioassays