[PhD Thesis Presentation_zoom] - Ms. Shivani Sathish

Presenter: Ms. Shivani Sathish

Supervisor: Professor Amy Shen

Unit: Micro/Bio/Nanofluidics Unit

Title: Surface-based Microfluidic Systems for Enhanced Biomarker Detection

Abstract:

Surface-based Microfluidic Systems for Enhanced Biomarker De-tection

The 21^st century has seen a surge in the development of point-of-care (POC) testing systems integrated with microfluidic bioassay devices, to address the need for reliable, fast, and user-friendly disease diagnostics at the patient’s bedside or in remote locations. These systems are designed to be able to detect diagnostic biomarkers from a small quantity of the patient’s fluid sample, e.g., blood plasma, urine, etc., by exploiting their innate nature to bind to specific receptor molecules coated on microfluidic device surfaces.

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. These features are directly influenced by the cumulative effect of several inter-related factors. This thesis ex-plores the collective influence of three major fundamental factors: (1) surface chemistry, and (2) biomarker transport and (3) biomolecular reactions at the microscale, to ulti-mately propose design principles for the development of rapid, sensitive and user-friendly fluorescence-based point-of-care (POC) disease diagnostic devices.

First, we exploited radio-frequency (RF) air plasma to covalently tether receptor pro-teins within polymethyl methacrylate (PMMA) microfluidic bioassay devices at high-throughput. We observed that 27 kJ of air plasma generated carboxyl (COOH)-rich PMMA surfaces with the highest affinity towards a wide range of receptor proteins, namely, immunoglobulin G (IgGs), streptavidin and major outer membrane proteins (MOMP) of Chlamydia trachomatis, when coupled with 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry, at a pH of 7.4.

Next, these PMMA microfluidic bioassay devices were integrated with a palm-sized modular Fluid Handling Device (FHD) that allowed precise mixing, filtration, and delivery of fluids to the microfluidic device for subsequent detection of C. trachomatis specific antibodies, with a limit of detection (LoD) of 7   nM within 15 mins, serving as a “proof-of-concept” manually operated point-of-care (POC) testing device.　
To investigate mass transport-dependent kinetic enhancements in microfluidic bioas-say systems, we developed novel and truly 3D glass microfluidic devices where the surfaces were first patterned with (3-aminopropyl) triethoxysilane (APTES) by microcontact printing for subsequent coupling with receptor molecules such as IgGs and DNA aptamers using standard EDC–NHS chemistry. By analyzing real-time binding events between varying concentrations of fluorescently-labelled ligand and receptor IgGs, we re-examined the scaling analyses reported in prior literature, and proposed two control parameters that are pivotal to achieving rapid and sensitive ligand detection in microfluidic devices. First, we defined a local Peclet number Peδ that characterized the balance between local convection and diffusion driven transport of ligands. We observed homogeneous binding of ligands across the receptor-coated surfaces at Peδ>>10⁵ with an added advantage of enhanced receptor–ligand reaction kinetics as a result of steady ligand replenishment. Additionally, we proposed the use of the kinetic Damkohler number (Da_kinetic) to characterize the balance between the rates of receptor–ligand binding and enhanced convection-driven ligand replenishment.

Finally, using a model receptor–ligand IgG reaction system, we demonstrated that rapid (detection time of 10 mins) and sensitive (LoD of 11.63 pM) ligand detection can be achieved in microfluidic devices with Peδ>>10⁷forDa_kinetic<<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 point-of-care testing systems in the near future.