Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry

Abstract

Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, we find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher absolute sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the observed signal gain of our cocaine sensor 10-fold. We demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.

Multi‐Level Logic Gate Operation Based on Amplified Aptasensor Performance

“…[14] Therefore we introduce the concept of electrochemical rectification (ECR) for signal amplification.…”

Aptamer Functionalized Microcantilever Sensors for Cocaine Detection

“…20,32 Baker et al 33 used the same DNA aptamer in electronic aptamer-based (E-AB) sensors and measured a dissociation constant (K d ) of 90 μM for cocaine/aptamer binding and detection limits of below 10 μM for cocaine molecules. White et al 34 investigated the influence of aptamer surface coverage on the performance of electrochemical aptamer-based sensor (E-AB cocaine sensor) in detection of cocaine molecules and reported that an increase in surface coverage resulted in lower response from E-AB sensors for similar levels of cocaine concentrations. They concluded that this phenomenon occurred due to unfavorable interaction arising between neighboring aptamers.…”

“…Thus, White et al 34 hypothesized that the optimal surface coverage for E-AB sensor will correspond to surface-immobilized aptamer spacing such that a single aptamer chain can undergo a conformational change with little or no interaction with neighboring molecules. However, the low affinity between cocaine and aptamer limited the detection threshold to 10 μM cocaine concentration.…”

“…20,32,34,41 In summary, the cocaine aptamer has been used in a variety of different platforms, but low affinity between the cocaine and aptamer molecules limits the detection threshold between 1 and 10 μM.…”

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

“…Run a square wave measurement from 0 to -0.6 V with an amplitude of 25 mV and a step size of 1mV. The optimal square wave frequency will depend on the details of the probe architecture 2,3 ; for the probe architectures we have employed the optimal values are typically in the range 60 to 600 Hz. You should see a rounded peak at approximately -0.35 V, the redox potential of methylene blue (the peak potential may shift slightly depending on the precise pH of your test solution).…”