טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentOstromohov Nadya
SubjectHigh Sensitivity Sequence-Specific Detection of Nucleic
Acids Using Isotachophoresis and Peptide Nucleic
Acid Probes on a Microfluidic Chip
DepartmentDepartment of Mechanical Engineering
Supervisor Professor Moran Bercovici
Full Thesis textFull thesis text - English Version


Abstract

Sensitive, sequence specific DNA detection plays a crucial role in various biosensing applications including medical diagnostics, medical and biological research, food and water safety and forensics. The use of amplification techniques such as polymerase chain reaction (PCR) is a common approach for improvement of sensitivity. However, PCR reactions suffer from an inherent amplification bias, and require significant sample preparation, and a well-controlled environment. This results in a growing need for simpler and faster alternatives to amplification. An amplification-free nucleic acids detection method able to reduce the typical assay time is of special importance in faster disease diagnosis and treatment.

We present a novel assay for rapid high sensitivity detection of nucleic acids without amplification using isotachophoresis and peptide nucleic acid (PNA) probes. Isotachophoresis (ITP) is an electrophoretic technique in which analytes of interest can be focused at the interface between two electrolytes characterized by high and low electrophoretic mobilities. Utilizing the neutral backbone of peptide nucleic acids, our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model which allows predicting the length of targets that could be focused, for a given ITP chemistry, and experimentally demonstrate detection of targets as short as 17 nt and a limit of detection of 100 fM with a dynamic range of 5 decades. The assay requires 15 min to complete, and can be potentially used for a wide range of applications where rapid and highly sensitive detection of nucleic acid biomarkers is required.