Stimuli-Responsive Templated Polymer as a Target Receptor for a Conformation-based Electrochemical Sensing Platform

Abstract

The use of highly crosslinked molecularly imprinted polymers as a synthetic target receptor has the limitations of restricted accessibility to the binding sites resulting in slow response time. Moreover, such artificial receptors often require additional transduction mechanisms to translate target binding events into measurable signals. Here, we propose the development of a single-chain stimuli-responsive templated polymer, without using any covalent interchain crosslinkers, as a target recognition element. The synthesized polymer chain exhibits preferential binding with the target molecule with which the polymer is templated. Moreover, upon specific target recognition, the polymer undergoes conformation change induced by its particular stimuli responsiveness, namely the target binding event. Such templated single-chain polymers can be attached to the electrode surface to implement a label-free electrochemical sensing platform. A target analyte, 4-nitrophenol (4-NP), was used as a template to synthesize a poly-N-isopropylacrylamide (PNIPAM)-based copolymer chain which was anchored to the electrode to be used as a selective receptor for 4-NP. The electrode surface chemistry analysis and the electrochemical impedance study reveal that the polymer concentration, the interchain interactions, and the Hofmeister effect play a major role in influencing the rate of polymer grafting as well as the morphology of the polymers grafted to the electrode. We also show that the specific binding between 4-NP and the copolymer results in a substantial change in the charge transfer kinetics at the electrode signifying the polymer conformation change.

Keywords: Conformation Change; Electrochemical Sensing; Molecular Imprinting; Nitrophenol; Poly(N-isopropylacrylamide); RAFT; Stimuli-Responsive; Templated Polymer.

Figure 1.

A conceptual illustration of the synthesis of a single-chain PNIPAM-based templated copolymer as a receptor for the target 4-nitrophenol (4-NP) and its use as a conformation-dependent electrochemical sensor.

Figure 2.

Fourier transform infrared (FTIR) spectra comparison between the PNIPAM-MAA-VP copolymer (blue) and the PNIPAM homopolymer (red). The overall FTIR for both samples are shown in (a) with select regions magnified in insets (b-d).

Figure 3.

Characterization of the temperature- and time-dependent polymer dispersion in solution. (a) Distribution of the hydrodynamic radius (D_h) of the polymers at different temperature measured via Dynamic Light Scattering (DLS) in MOPS buffer (pH 7). Legends:15°C (purple), 25°C (green), 35°C (blue), 45°C (red), 55°C (black); (b) The average diameter of the polymer sample as a function of temperature. The diameter is taken at the maximum volume percentage point from the DLS measurements. The vertical line indicates the lower critical solution temperature (LCST). Measurements were repeated three times (n = 3); (c) Nyquist plot of the polymer-attached gold electrode as a function of time; (d) Plot of charge transfer resistance (R_ct) as a function of time (n = 3).

Figure 4.

The XPS spectra for S2p (a-d) and N1s (e-h) of gold electrodes coated with PNIPAM-MAA-VP copolymers with two different grafting times (20 seconds & 30 minutes) with and without TCEP. The S2p spectra analyzes the condition of the thiol bond formed at the polymer-gold interface. Legends for the S2p Spectra: S₀: 160.7–161eV (wine); S₁: 162.6–162.9eV (dark cyan); S₂: 163.7–164.2eV (purple); S₃: 165.2eV (pink); S₄: 165.6–166.2eV (olive); S₅: 168eV (dark gray). The N1s spectra mainly emphasizes the functional moieties of PNIPAM and VP. Legends for the N1s Spectra: N₀: 392.5eV (dark gray); N₁: 395.1eV (orange); N₂: 395.8–396.4eV (green); N₃: 396.5–397.5eV (magenta); N₄: 398.2–398.7eV (violet); N₅: 400.3eV (gray). Each figure contains real data (blue hollow circle), fitted data (red curve), and background (black curve). The vertical lines indicate the position of the specific peaks.

Figure 5.

Grafting of the synthesized copolymer chain to a gold electrode. In the presence of TCEP, DDMAT-terminated copolymer (a) can be converted into a thiol-terminated copolymer (b). The polymers that are grafted to the electrode surface include both DDMAT-terminated (a & a’) and thiol-terminated (b) copolymers. There is also a possible attachment of dodecane groups (c) that have been cleaved from DDMAT assisted by TCEP.

Figure 6.

The electrode surface coverage characterization using EIS for two different polymer concentrations, (a) 0.05 μg/mL and (b) 0.5 μg/mL, with varying polymer grafting times. The inset in (a) shows the circuit fit for the Nyquist plots for both polymer concentrations. (c) shows the surface coverage θ(t) plot for the grafted polymer as a function of time for both polymer concentrations. (d) shows the change in the apparent rate constant (K_app) as a function of time. The time-dependent rate constant profiles follow the two-phase exponential decay model. Legends for (a) & (b): Bare gold (black); 30 sec (red); 1 min (green); 2 mins (blue); 4 mins (orange); 8 mins (magenta); 16 mins (brown); 32 mins (violet); fitted data (grey). Legends for (c) & (d): 0.05 μg/mL (red); fitted curve for 0.05 μg/mL (Red dotted line); 0.5 μg/mL (blue); fitted curve for 0.5 μg/mL (blue dotted line).

Figure 7.

Target binding characterization for the electrochemical sensing of 4-NP. The Nyquist plots after exposing the polymer-modified electrode to varying concentrations of 3-NP (a, b) and 4-NP (d, e). The binding of the template (4-NP) results in a change in the charge transfer kinetics at the polymer-gold interface. The plots in (b) and (e) show the changes in the cyclic voltammetry (CV) when exposed to 3-NP and 4-NP, respectively. The plot in (c) shows the difference in the apparent rate constant (K_app) upon specific binding of 4-NP (red) compared to 3-NP (blue) and 2-NP (green). The plot in (f) shows the change in the charge transfer resistance (ΔR_ct/R_ct,0) of the templated polymer versus target (4-NP) concentration. In the case of 3-NP and 2-NP, the R_ct change and K_app do not show any significant trend as a function of concentration. For each experiment, the nitrophenol sample (2-, 3-, or 4-NP) was exposed for 1 minute to the electrode prior to electrochemical measurements. Legends for (a, b, d, e): 0 μM (black); 1 μM (blue); 5 μM (red); 10 μM (green); 15 μM (brown); 20 μM (orange). The arrows in (b) and (e) represent forward and reverse directions of CV.

Figure 8.

Selective binding of 4-NP assessed via electrochemical sensing on the river water. (a) The Nyquist plot for the sensor when exposed to varying concentrations of 4-NP in the presence of a mixture of interferents (20 μM each for Phenol, 2-Chlorophenol, and 2-Aminophenol); (b) The Nyquist plot for varying concentrations of 4-NP without any interferents added; (c) The Nyquist plot for varying concentrations of interferents (same concentration for all three species); (d) The calibration curve showing the change in the charge transfer resistance (ΔR_ct/R_ct,0) versus 4-NP concentrations. Legends for (a – c): 0 μM (black); 1 μM (blue); 5 μM (red); 10 μM (green); 15 μM (brown); 20 μM (orange). Legends for (d): 4-NP only (black), 4-NP with interferents (red), and interferents only (blue).