טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZeidman Kalman Tal
SubjectOn-Chip Isotachophoresis for Biosensing Applications
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor Professor Moran Bercovici


Abstract

Nucleic Acid (NA) and proteins are the main biomarkers of interest in diagnostics. Sensitive and specific detection of NA and proteins is of particular importance in diagnostics, with applications including parental testing and identification of genetic diseases, detection of bacterial and viral infections, and cancer screening and monitoring.

Isotachophoresis (ITP) is an electrophoretic technique in which analytes of interest are focused at the interface of two distinct electrolyte solutions characterized by high and low electrophoretic mobility. ITP has been used for separation, extraction and concentration of various of charged analytes and for reactions acceleration.

This work presents two novel assays leveraging on-chip isotachophoresis for high sensitivity biosensing applications.

The first assay is a single-step homogenous (free solution) assay that uses Morpholino non-focusing probes in continuous-injection ITP that gives rise to unique accumulation-dissociation dynamics, which can be leveraged to achieve highly specific detection in the presence of a high concentration of mismatched targets (“mismatches”). We show that assay conditions can be selected such that, on experimental timescales, complementary targets (“matches”) yield a linear increase in signal, while mismatches exhibit a non-monotonic signal related to their dissociation rate from the probe. We demonstrate that for a given analysis time, specificity is limited by the sensitivity of the system. Our standard CCD-based optical system show a limit of detection (LoD) of ~100 pM, allowing demonstration of 1000:1 specificity for 4/25 bp sequences, and 10:1 specificity for 20/25 bp sequences. Significant improvement in specificity will likely be possible using high-numerical aperture confocal imaging. Moreover, we present an analytical prediction of the assay’s specificity and show that for a known mismatch to match concentration ratio, the koff value of the mismatch can be used to extract the minimal time at which the match could be distinguished from the mismatch with a 3:1 signal ratio.

The second assay is a new class of ITP heterogeneous (surface-based) immunoassay for the detection of a bacterial antigen, protective antigen (PA) - a component of Bacillus anthracsis toxins (infection with B. anthracsis spores induces an acute anthrax disease).  We use ITP to focus and deliver a high concentration target to a surface formed by paramagnetic beads pre-functionalized with capture antibodies, thus we accelerate antigen-antibody reaction. We optimize the ITP-based immunoassay and demonstrate ~1000-fold improvement in LoD compared to a standard continuous flow based immunoassay.

Furthermore, we demonstrated the capability of leveraging ITP to focus and perform direct analysis of NA and protein in urine samples.