Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors

Abstract

Electrochemical aptamer-based (E-AB) sensors are a versatile sensing platform that can achieve rapid and robust target detection in complex matrices. However, the limited sensitivity of these sensors has impeded their translation from proof-of-concept to commercial products. Surface-bound aptamers must be sufficiently spaced to bind targets and subsequently fold for signal transduction. We hypothesized that electrodes fabricated using conventional methods result in sensing surfaces where only a fraction of aptamers are appropriately spaced to actively respond to the target. As an alternative, we presented a novel aptamer immobilization approach that favors sufficient spacing between aptamers at the microscale to achieve optimal target binding, folding, and signal transduction. We first demonstrated that immobilizing aptamers in their target-bound, folded state on gold electrode surfaces yields an aptamer monolayer that supports greater sensitivity and higher signal-to-noise ratio than traditionally prepared E-AB sensors. We also showed that performing aptamer immobilization under low ionic strength conditions rather than conventional high ionic strength buffer greatly improves E-AB sensor performance. We successfully tested our approach with three different small-molecule-binding aptamers, demonstrating its generalizability. On the basis of these results, we believe our electrode fabrication approach will accelerate development of high-performance sensors with the sensitivity required for real-world analytical applications.

Keywords: aptamer; bundling effect; electrochemistry; sensing; small molecules; target-assisted aptamer immobilization.

Figure 1.

E-AB sensor performance using gold electrodes modified with ADE-25-MB either alone or bound to adenosine. (A) Modification of an electrode using either the traditional immobilization protocol (left) or our target-assisted immobilization strategy (right). Square-wave voltammetry (SWV) spectra of electrodes modified with the aptamer-target complex (left) or aptamer alone (middle) in (B) buffer or (C) 50% serum and corresponding calibration curves and linear ranges (right) collected using electrode prepared with target-assisted aptamer immobilization approach (red) or the traditional method (black). Error bars represent the standard deviation for three working electrodes from each measurement.

Figure 2.

E-AB sensor performance using electrodes modified with COC-32-MB via target-assisted immobilization. SWV spectra collected at various concentrations of cocaine using electrodes modified in high-salt PBS with (A) 2 mM or (B) 250 μM cocaine. (C) Calibration curves derived from SWV spectra for electrodes modified with COC-32-MB plus 2 mM (brown) or 250 μM cocaine (pink) and electrodes modified with aptamer alone (black). (D) Linear ranges and LODs of electrodes fabricated via different methods. Error bars represent the standard deviation for three working electrodes from each measurement.

Figure 3.

E-AB sensor performance using gold electrodes modified with target-bound COC-32-MB. SWV spectra at various concentrations of cocaine from electrodes modified with COC-32-MB plus cocaine in low-salt (A) PBS or (B) Tris. (C) Calibration curves derived from the SWV spectra shown in panel A (blue) and panel B (red) or from electrodes modified in cocaine-containing high-salt PBS (brown). Detection of cocaine in 50% saliva using electrodes modified with (D) aptamer-target complexes in low-salt Tris or (E) aptamer alone in high-salt PBS. (F) Calibration curves derived from the SWV spectra shown in panels D (red) and E (black). (G) Linear ranges and LODs of electrodes fabricated via different methods in buffer or 50% saliva. Error bars represent the standard deviation for three working electrodes from each measurement.

Figure 4.

E-AB sensor performance using electrodes fabricated with SC-34-MB. (A) Effect of MDPV concentration on surface coverage during electrode modification. (B) Signal gain for the various electrodes prepared in panel A from various concentrations of MDPV in buffer. (C) Detection of MDPV in 50% urine using modified electrodes prepared in low-salt Tris with SC-34-MB alone (M1) or in the presence of 50 μM MDPV (M4). SWV spectra from various concentrations of MDPV using electrodes modified via target-assisted immobilization in either (D) low-salt or (E) high-salt PBS. (F) Calibration curves derived from the SWV spectra shown in D (red) and E (brown) or from electrodes prepared in low-salt (blue) or high-salt (navy) Tris buffer. (G) Linear ranges and LODs of electrodes fabricated via different methods in buffer or 50% urine. Error bars represent the standard deviation for three working electrodes from each measurement.

Figure 5.

E-AB sensor performance using electrodes modified with SC-34-MB in the presence (target-assisted immobilization) or absence (traditional immobilization) of 50 μM MDPV in low-salt PBS at pH (A) 8.0, (B) 7.4, (C) 7.0, and (D) 6.0. The top panels show calibration curves for electrodes fabricated via target-assisted aptamer immobilization (red) or traditional modification method (black) produced by challenging with 0–100 μM MDPV, and bottom panels show their respective linear ranges. Error bars represent the standard deviation of measurements from three independently fabricated electrodes.