Study of surface modification strategies to create glassy carbon-supported, aptamer-based sensors for continuous molecular monitoring

Abstract

Electrochemical, aptamer-based (E-AB) sensors uniquely enable reagentless, reversible, and continuous molecular monitoring in biological fluids. Because of this ability, E-AB sensors have been proposed for therapeutic drug monitoring. However, to achieve translation from the bench to the clinic, E-AB sensors should ideally operate reliably and continuously for periods of days. Instead, because these sensors are typically fabricated on gold surfaces via self-assembly of alkanethiols that are prone to desorption from electrode surfaces, they undergo significant signal losses in just hours. To overcome this problem, our group is attempting to migrate E-AB sensor interfaces away from thiol-on-gold assembly towards stronger covalent bonds. Here, we explore the modification of carbon electrodes as an alternative substrate for E-AB sensors. We investigated three strategies to functionalize carbon surfaces: (I) anodization to generate surface carboxylic groups, (II) electrografting of arenediazonium ions, and (III) electrografting of primary aliphatic amines. Our results indicate that electrografting of primary aliphatic amines is the only strategy achieving monolayer organization and packing densities closely comparable to those obtained by alkanethiols on gold. In addition, the resulting monolayers enable covalent tethering of DNA aptamers and support electrochemical sensing of small molecule targets or complimentary DNA strands. These monolayers also achieve superior stability under continuous voltammetric interrogation in biological fluids relative to benchmark thiol-on-gold monolayers when a positive voltage scan window is used. Based on these results, we postulate the electrografting of primary aliphatic amines as a path forward to develop carbon-supported E-AB sensors with increased operational stability. Graphical abstract

Explaining the Decay of Nucleic Acid-Based Sensors under Continuous Voltammetric Interrogation

“…Our strategy to study NBE stability is based on serially interrogating sensors via cyclic and square wave voltammetry in media of varying compositions to independently evaluate mechanisms of signal degradation (Figure B–E) . We use cyclic voltammetry to track changes in the oxygen reduction and capacitive currents resulting from the loss and/or reorganization of blocking monolayer elements on the electrode surface . As a complementary technique, we use square wave voltammetry to monitor with high sensitivity changes in electron transfer originating from the loss of reporter-modified oligos from the electrode surface .…”

Os(ii/iii) complex supports pH-insensitive electrochemical DNA-based sensing with superior operational stability than the benchmark methylene blue reporter

“…S2 and S3, ESI † ). To evaluate the performance of this complex relative to benchmark MB, we employed the commercially available analog new methylene blue (NMB), 13 which has two amines available for coupling reactions.…”

“…Seeking to address the first problem, several studies have focused on different elements of the sensing layer like the chemical nature of the blocking alkylthiol 8,9 or the DNA strands, 10 the technique used for sensor interrogation, 11,12 and the electrode material. 13 However, the second problem has received significantly less attention. Seventeen years after the first report on this technology, 3 most published E-AB sensors still rely on the use of a single redox reporter which undergoes pH-dependent electron transfer: methylene blue (MB).…”

Continuous Square Wave Voltammetry for High Information Content Interrogation of Conformation Switching Sensors

“…When the same 6 frequencies are isolated from the native 50 Hz sweep, we find that the signal change upon target addition matches with what is observed with SWV (Figure bottom). Plotting signal changes at every collected δ t , and thus frequency, gives a highly resolved view of the frequency response of the respective E-AB sensor . The most remarkable percent signal change for ATP sensors is observed at around 175% given at 25,000 Hz, and overall higher percent signal changes (>100%) are observed at higher frequencies (>500 Hz).…”

“…Conversely, two voltammetric sweeps of cSWV taken at a native frequency of 50 Hz with and without a target yield 1000 voltammograms between 50 Hz and 50 kHz (Figure 5 Plotting signal changes at every collected δt, and thus frequency, gives a highly resolved view of the frequency response of the respective E-AB sensor. 23 The most remarkable percent signal change for ATP sensors is observed at around 175% given at 25,000 Hz, and overall higher percent signal changes (>100%) are observed at higher frequencies (>500 Hz). Similarly, tobramycin sensors provided the highest percent signal changes, around 50%, and both signal on and off responses were observed as expected from tobramycin sensors.…”