Magnetic particle based sensing platform for oligonucleotide and PCR amplicon detection

Abstract

In this thesis, magnetic particle (MP) based sensing platform for oligonucleotide and PCR amplicon detection is reported. In the first approach, MPs served as separation tools. The binding between biotin-labeled DNA hybrids and streptavidin-coated MPs was achieved through the biotin-streptavidin affinity reaction and resulted in the formation of DNA-particle conjugates. These conjugates were then separated from the solution through magnetic separation process, and the signal changes in the supernatant were monitored by fluorescence and electrochemical measurements. The fluorescence detection part gave a proof-of-concept of the MP-based DNA detection approach, and a fluorescent label fluorescein was employed. The following content was based on the electrochemical detection, an electro-active intercalator anthraquinonemonosulfonic acid (AQMS), which could preferentially bind with double-strand DNA (dsDNA) was used. Differential pulse voltammetry (DPV) was employed to monitor the electrochemical response in the supernatant. In the second approach, an enzyme-labeled electrochemical PCR amplicon detection platform was developed. Here, MPs were used both as the separation tools and the matrix for biomolecule immobilization. The biotin and an antigen digoxigenin (DIG) dually-labeled PCR amplicons were sequentially reacted with streptavidin-coated MPs and the anti-DIG modified horseradish peroxidase (HRP) to form protein-DNA-particle complexes. Then, substrate solution which contained 3,3`,5,5` -tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) were added for enzyme-substrate reaction. After incubation, this mixture was attracted and confined onto the electrode, and electrochemical measurements were performed on the particle surface. The mechanism of this electrochemistry based measurement is that, TMB could be consumed in the enzymatic reaction; besides, TMB could also be electrochemically oxidized on the electrode surface. After enzymatic reaction, the remaining TMB would produce electrochemical signal, and the changes of the oxidation peak currents could be used for quantification of the enzymatic reaction, therefore could indicate the amplification of PCR amplicons. DPV was used for monitoring the electrochemical response.