טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBelenkovich Merav
SubjectAccelerated Nucleic Acid Hybridization on Surface
Based Biosensors Using Isotachophoresis
DepartmentDepartment of Mechanical Engineering
Supervisor Professor Moran Bercovici
Full Thesis textFull thesis text - English Version


Abstract

Highly sensitive, specific and rapid detection of biomarkers is the cornerstone of point-of-care diagnostics, with surface-based biosensors comprising the majority of such sensors.  Surface biosensors are based on a chemical reaction between target biomolecules in the sample and matching receptors on the surface. Regardless of the specific transduction mechanism used for detecting the reaction, the sensitivity of all such biosensors is fundamentally limited by the rate at which target molecules bind to the surface. This rate is proportional to the concentration of the target molecule, and becomes the limiting factor in detecting low concentrations.

In the first part of the dissertation, we present a novel method for acceleration of surface-based reactions using isotachophoresis (ITP).  ITP is an electrophoretic technique in which analytes of interest can be focused at the interface of two distinct electrolyte solutions characterized by high and low electrophoretic mobilities.  We use ITP to focus a sample of interest and deliver a high concentration target to a pre‑functionalized surface, thus enabling rapid reaction at the sensor site.  The concentration of the focused analyte is bound in space by the ITP interface, and upon reaction with the surface continues electromigrating downstream removing any contaminations or reacted sample molecules from the surface. We designed a novel microfluidic chip where reaction surfaces are formed by paramagnetic beads, immobilized at desired sites by an external magnetic field. Using this chip, we compared ITP-based surface hybridization to standard continuous flow-based hybridization and experimentally demonstrated a two orders of magnitude improvement in limit of detection of a 3 min nucleic acid hybridization assay.  

In the second part of the dissertation we present a new method for accurate detection of sample location in peak mode ITP, and its delivery to a designated reaction site.  In this technique we detect the passage of the ITP interface through a microchannel with multiple constrictions, by simply monitoring the electric current across the channel, which exhibits sharp decreases as the ITP interface moves more rapidly through the higher current density constrictions.  We show that cross-correlation between the electric current signal and a pre-defined step function is an effective method for detecting changes in the slope of the electric current curve in real-time, and is robust to changes in the composition of the buffers. We demonstrate the use of the technique to accurately deliver sample to a designated location in the channel with an accuracy of 50 μm.