Codeposition Enhances the Performance of Electrochemical Aptamer-Based Sensors

Abstract

Electrochemical aptamer-based (EAB) sensors, a minimally invasive means of performing high-frequency, real-time measurement of drugs and biomarkers in situ in the body, have traditionally been fabricated by depositing their target-recognizing aptamer onto an interrogating gold electrode using a "sequential" two-step method involving deposition of the thiol-modified oligonucleotide (typically for 1 h) followed by incubation in mercaptohexanol solution (typically overnight) to complete the formation of a stable, self-assembled monolayer. Here we use EAB sensors targeting vancomycin, tryptophan, and phenylalanine to show that "codeposition", a less commonly employed EAB fabrication method in which the thiol-modified aptamer and the mercaptohexanol diluent are deposited on the electrode simultaneously and for as little as 1 h, improves the signal gain (relative change in signal upon the addition of high concentrations of the target) of the vancomycin and tryptophan sensors without significantly reducing their stability. In contrast, the gain of the phenylalanine sensor is effectively identical irrespective of the fabrication approach employed. This sensor, however, appears to employ binding-induced displacement of the redox reporter rather than binding-induced folding as its signal transduction mechanism, suggesting in turn a mechanism for the improvement observed for the other two sensors. Codeposition thus not only provides a more convenient means of fabricating EAB sensors but also can improve their performance.

Figure 1.

EAB sensors have most often been fabricated using a “sequential deposition” in which a gold electrode is exposed to a solution of thiol-modified aptamer followed by an overnight “backfilling” incubation in dilute mercaptohexanol (MCH). Here we compare the advantages of this approach via a “codeposition” approach in which only a mixed solution of mercaptohexanol and a thiol-modified aptamer is employed. Although the latter has seen widespread use in the fabrication of DNA-modified monolayers, it has rarely been employed in the fabrication of sensors of this specific type.

Figure 2.

Codeposition often improves EAB sensor gain, which is the relative signal change from no target to high target concentrations. (Top row) Shown are binding curves for (A) vancomycin-, (B) tryptophan-, and (C) phenylalanine-detecting EAB sensors when the sensors are fabricated using sequential deposition (500 nM aptamer followed by 10 mM MCH) or codeposition (500 nM aptamer at the indicated MCH concentration). In each case, codeposition at 10 or 100 μM MCH gives rise to gain that matches or improves on the gain seen for sensors fabricated using sequential deposition. In contrast, at higher or lower MCH concentrations, the gains were obtained using codeposition fall. (D, E, F) The poorer gain seen at lower MCH concentrations is associated with voltammogram baselines that slant strongly upward, suggesting that oxygen reduction is occurring as a result of incomplete monolayer formation (illustrated in panel G). In contrast, the methylene blue peak is suppressed upon codeposition at higher MCH, presumably because fewer aptamers are immobilized under these conditions. Only at intermediate MCH concentrations are these problems avoided to create high-performance sensors. The data presented here were collected from sensors fabricated via overnight deposition for codeposition and 1 h aptamer deposition followed by overnight MCH deposition for sequential deposition.

Figure 3.

(A–C) The signaling properties of sensors fabricated via codeposition are only a weak function of the concentration of DNA employed during deposition. Here we employed overnight codeposition.

Figure 4.

Signal gains (e.g., where the fitted Langmuir isotherms hit the right side of these plots) of our (A) vancomycin- and (B) tryptophan-detecting EAB sensors increase when they are fabricated in the presence of their target. This effect occurs for both sequential and codeposition and is additive with the improvements obtained via codeposition. (C) In contrast, the gain of our phenylalanine-detecting sensor varies little across these fabrication methods. The data presented here reflect sensors fabricated using overnight deposition for both codeposition and sequential deposition (i.e., in the MCH backfill solution overnight). We used overnight sequential deposition as the performance of sensors fabricated using this longer deposition time is improved relative to sensors fabricated using shorter incubations (Figure S1).

Figure 5.

Circular dichroism spectra of the vancomycin-binding and tryptophan-binding aptamers change dramatically between their target-bound and unbound statues (A, B), suggesting that target binding induces a large conformation change. (C) In contrast, the phenylalanine-binding aptamer shows no such change, suggesting that it remains folded even in the absence of a target.

Figure 6.

Signaling and drift properties of EAB sensors are independent of the duration of the codeposition (deposition times indicated). Indicated in each panel are the codeposition times we employed.