Currently, there is a need for reliable biosensors to study biological systems in order to answer important biochemical questions and to provide real-time therapeutic monitoring in order to improve patient outcome. This summer, I have been preparing and testing electrochemical aptamer-based (E-AB) biosensors in Dr. Ryan White’s research laboratory with the help of Dr. Florika “Flaire” Macazo and Ph.D student, Mirelis Santos Cancel. E-AB biosensors are sensors that are modified with redox-labeled, conformation-changing aptamers that can be used for reagent-less electrochemical measurements. These aptamers are specific nucleic acid sequences (RNA or DNA) with a redox marker, generally methylene blue, on the distal end of the aptamer that participates in electron transfer with the electrode surface. These aptamers bind specifically to target proteins or molecules in solution and undergo a conformational change that results in a change in electron transfer between the redox marker and the electrode surface, as shown in the figure below. This change in electron transfer can be detected and measured directly as the concentration of analyte is varied.
NSF REU student Mai Lam (second from left) with (from left) UMBC graduate student Steven Lowery, Dr. Ryan White, UMBC graduate student Mirelis Santos Cancel, UMBC graduate student Daniel Kazal, UMBC Ph.D graduate Florika “Flaire” Macazo, UMBC summer intern William Dean, UMBC undergraduate student Inayah Entzminger and UMBC undergraduate student Nicholas Vaccaro.
Aptamer conformational change upon binding of target stimulus.
My project in Dr. White’s laboratory this summer involves preparing various E-AB biosensors modified with various DNA and RNA aptamer sequences and using these biosensors to characterize gentamycin sulfate (structure shown below). Gentamycin sulfate is a cell culture antibiotic that can be used for long-term virus and tissue culture studies. To prepare the E-AB biosensors for electrochemical measurements, I have to mechanically polish, electrochemically clean and modify gold macro-electrodes with specific aptamers. Thus far, I have been working with the Parent RNA Tobramycin and a specially modified DNA Tobramycin 1 aptamer sequence (structures shown below).
Parent RNA Tobramycin Aptamer Sequence
Modified DNA Tobramycin 1Aptamer Sequence
I have been characterizing the E-AB biosensors against gentamycin sulfate through a method of electrochemical measurement known as square wave voltammetry. In order to conduct the electrochemical measurements, titrations of gentamycin sulfate were done with the E-AB biosensors, in which specific amounts of a stock solution of gentamycin sulfate was added to buffer for detection by the E-AB biosensor. The RNA Tobramycin aptamer undergoes a specific conformational change when the target protein (tobramycin or gentamycin sulfate) is added to solution, and this conformational change is illustrated below. The extent of the conformational change of the RNA Tobramycin aptamer is dependent upon of the frequency of the signal on the E-AB biosensor, and this phenomenon is also illustrated in the figure below.
Left: conformational change of RNA Tobramycin aptamer as it binds tobramycin at lower frequencies. Right: conformational change of RNA Tobramycin aptamer as it binds tobramycin at higher frequencies.
The frequency at which a titration occurs can determine whether an E-AB sensor displays a ‘signal-on’ or ‘signal-off’ trend. In a ‘signal-on’ trend, the addition of the target protein results in a conformational change in the aptamer that increases the amount of electron transfer between methylene blue, the redox marker, and the electrode surface. Conversely, in a ‘signal-off’ trend, the addition of the target protein results in a conformational change in the aptamer that decreases the amount of electron transfer between methylene blue and the electrode surface. In order to determine which frequencies were optimal for the Parent RNA Tobramycin aptamer and unknown DNA Tobramycin aptamer sequences, frequency sweeps were conducted at various frequencies to determine which frequencies resulted in the greatest amount of signal change. I determined that the optimal frequency for the Parent RNA Tobramycin aptamer against gentamycin sulfate was 30 Hz, while the optimal frequency for the unknown DNA Tobramycin aptamer.
The various aptamers were titrated with gentamycin sulfate at various concentrations and the signal change that occurred was measured using an electrochemical technique known as square wave voltammetry (SWV). It was experimentally determined that a titration with gentamycin sulfate completed for the Parent RNA Tobramycin aptamer resulted in a signal-off trend, while a titration with gentamycin sulfate completed for the specially modified DNA Tobramycin 1 aptamer resulted in a signal-on trend. These results can be seen below.
Left: SWV for Parent RNA Tobramycin Aptamer at 30 Hz without gentamycin sulfate (red) and with final concentration of 2.5 mM gentamycin sulfate (blue). Right: SWV for Modified DNA Tobramycin 1 Aptamer at 700 Hz without gentamycin sulfate (red) and with final concentration of 3.2 mM gentamycin sulfate (blue).
Left: Titration curve with equation for Parent RNA Tobramycin Aptamer at 30 Hz with additions of gentamycin sulfate. Right: Titration curve with equation for Modified DNA Tobramycin 1 Aptamer at 700 Hz with additions of gentamycin sulfate.
The next step for this project would be to further characterize the specially modified DNA Tobramycin 1 aptamer with gentamycin by conducting titrations with gentamycin sulfate in fetal bovine serum. Following this, full characterizations of other specially modified DNA Tobramycin aptamers with gentamycin sulfate will be conducted in buffer and fetal bovine serum.