Aptamer Sensors for Drugs of Abuse and Medical Biomarkers: Design, Engineering and Application in Complex Samples

Authors

Daniel Roncancio, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Chemistry

First Advisor's Name

Yi Xiao

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Anthony DeCaprio

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Anthony McGoron

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Alexander Mebel

Fourth Advisor's Committee Title

Committee member

Fifth Advisor's Name

Kevin O'shea

Fifth Advisor's Committee Title

Committee member

Keywords

aptamer, sensor, electrochemistry, cocaine, adenosine, exonuclease, MDPV, small molecules, serum

Date of Defense

6-22-2018

Abstract

Aptamers are short oligonucleotide sequences (DNA or RNA) capable of high affinity and specific binding to a molecule or a family of molecules. Aptamers are lower in cost and exhibit higher reproducibility when compared to antibodies and thus are well-suited for recognition and detection of small molecular targets such as drugs of abuse and small medical biomarkers. While aptamers have been extensively utilized for development of small molecule sensors, several limitations prevent measurements of complex or real-world samples. This dissertation describes methods, technologies, and assays that were developed with the goal of producing and/or improving aptamer-based sensors for target detection in complex samples. Aptamer engineering is detailed as an important facet of maximizing aptamer-sensor sensitivity and specificity, along with adaptation to various read-out mechanisms for improved selectivity. In chapter 3, an aptamer vii sensor for cocaine is developed based on binding between the fluorophore ATMND to the cocaine aptamer which results in quenching (i.e., ‘turn-off’) of the fluorescence of ATMND. Cocaine binding results in displacement of the ATMND and recovery of the fluorescence signal. Detection of cocaine is demonstrated with an engineered cocaine aptamer with higher affinity for cocaine, permitting over a 50-fold increase in sensitivity over other aptamer-based sensors. The method can be used in dilute biological fluids (e.g., saliva) with a single step reaction (seconds) and robust signal output. In chapter 4, a new adenosine specific aptamer is identified through rational engineering of a previously reported ATP-binding aptamer. The new adenosine aptamer is utilized to develop an electrochemical sensor for detection of adenosine in undiluted serum. The method displays 40-fold higher sensitivity in undiluted serum measurements over previously reported aptamer-based sensors for adenosine but also demonstrates specificity for adenosine over ATP, ADP and AMP that has not been previously reported. In chapter 5, a nuclease-guided truncation method is developed to yield optimal structure-switching aptamer sequences for the emergent illicit drug methylenedioxypyrovalerone (MDPV) and medical biomarkers ATP and deoxycorticosterone 21-glucoside (DOG). The method intelligently removes unessential nucleotides, producing truncated aptamer sequences with structure-switching functionality. This technique will be immediately useful for simple and low-cost development of aptamer-based electrochemical sensors.

Identifier

FIDC006889

Recommended Citation

Roncancio, Daniel, "Aptamer Sensors for Drugs of Abuse and Medical Biomarkers: Design, Engineering and Application in Complex Samples" (2018). FIU Electronic Theses and Dissertations. 3826.
https://digitalcommons.fiu.edu/etd/3826