טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentOstromohov Nadya
SubjectMicro-Scale Flow Confinement on Surfaces for Bioanalytical
Applications using Pressure- and Electric-
Actuation
DepartmentDepartment of Mechanical Engineering
Supervisors Professor Moran Bercovici
Dr. Govind Kaigala
Full Thesis textFull thesis text - English Version


Abstract

Methods allowing to spatially confine liquids and control their interaction with surfaces have gained significant attention in recent years due to their potential for advanced and precise processing of substrates, with applications ranging from fabrication of new materials by additive patterning, through stimulation of biological samples, to extraction of molecules for forensic and medical diagnostics applications. However, these techniques are not yet widely used for analysis of samples in standard laboratories due to their high level of complexity, cost, and the lack of integration of versatile analysis capabilities.

This dissertation is concerned with the theoretical analysis and experimental demonstration of new technologies for highly localized analysis of surface-based samples, and their implementation toward bioanalytical applications having an improved level of accuracy, control of reaction conditions, and range of applications.

In the first part of the dissertation, we present a method for sequential delivery of reagents to a reaction site with minimal dispersion of their interfaces. Using segmented flow to encapsulate the reagents as droplets while transmitted to the reaction-surface, dispersion between reagent plugs remains confined in a limited volume. In proximity to the surface, we use a passive microstructure to remove the oil phase such that the original reagent sequence is reconstructed, and only the aqueous phase reaches the reaction-surface. We analyze the conditions under which the method is applicable and implement it using a vertical microfluidic probe (vMFP), allowing contact-free interaction with biological samples. We demonstrate two assays: measurements of receptor-ligand reaction kinetics and of the fluorescence response of immobilized GFP to local variations in pH.

In the second part, we present a novel method for real-time monitoring and kinetic analysis of fluorescence in situ hybridization (FISH). We implement the method using a vMFP containing a microstructure designed for rapid switching between probe solution and nonfluorescent imaging buffer. The FISH signal is monitored in real-time during the imaging buffer wash, during which signal associated with unbound probes is removed. We provide a theoretical description and implement the method for characterization of FISH kinetics under conditions of varying probe concentration, destabilizing agent content, volume-exclusion agent content, and ionic strength. We show that our method can be used to investigate the effect of each of these variables, providing insight into processes affecting in situ hybridization.

In the last part, we present theoretical analysis and experimental demonstration of a new non-contact scanning probe, in which transport of fluid and molecules is controlled by electric fields. The electrokinetic scanning probe (ESP) enables local biochemical interactions with surfaces in liquid environments. We demonstrate the compatibility of the probe with a wide range of solutions and pH values, and its applicability for surface patterning, localized heating and 250-fold analyte stacking. Finally, we demonstrate integration of the electronics actuating the ESP into a self-contained compact device.  The ability to electrically control the transport of ionic-species and ?fluids to- and from- confined regions on substrates under well-controlled conditions in liquid environments opens the door to a range of new applications in deposition and extraction of biomolecules.