A Novel Target Receptor Design and Biosensing Platform Using RAFT Polymer-Based Molecular Templating
Authors: Si Bo , Yang Rongfang , Dutta Gaurab , Csoros John , Ahmad Habib M.N. , Seitz William Rudolf , Song Edward 
Published: February 2020

In this work, we propose a novel biosensing platform based on a single-chain molecularly templated polymer. We have utilized reversible addition-fragmentation chain transfer (RAFT) technique to synthesize a polymer-based target receptor consisting of N-isopropylacrylamide (NIPAM) as a backbone and methacrylic acid (MAA) and 4-vinylpyridine (4-VP) as functional monomers. In addition, vinylferrocene (VF) was incorporated into the polymer as a redox reporter. 4-nitrophenol (4-NP) was chosen as a model target for templating. The sensing platform was implemented by immobilizing the polymers on gold electrode surface, forming a brush-like polymer layer. As the target molecules bind to the polymer receptor, the polymer undergoes conformational change which can be detected via electrochemical methods. The proposed sensing platform is aimed to detect small species such as neurotransmitters and neurotoxins with high specificity.
One of the major challenges with biochemical sensor is achieving selectivity in analyte detection and therefore, a highly selective target receptor is needed [1]. One strategy to achieving this goal is the technique known as molecular templating or molecularly imprinted polymers (MIP) [2] where the polymer receptors are synthesized in the presence of the ‘template’ molecule to capture its chemical and structural motifs. Traditionally, the molecularly imprinted polymers were highly crosslinked 3-dimensional structures with the target binding sites embedded within the bulk polymer. However, the challenge with this architecture is that the response time is slow (due to the analyte having to penetrate the polymer layer to find the receptors) and removing the bound target species is difficult, rendering this approach not suitable for real-time continuous monitoring applications. In our approach, we propose a single-chain templated polymer-based target receptor as a recognition element for chemical detection. We have chosen 4-nitrophenol (4-NP), one of the priority toxicants according to the US Environmental Protection Agency, as our model target analyte.
The process for preparing the templated polymers is shown in Figure 1. Briefly, N-isopropylacrylamide (NIPAM), functional monomers, redox label, RAFT agent, initiator and the template monomers were added in a solvent for molecule assembly. Next, after removing the dissolved oxygen inside the mixed solution, the polymerization was initiated. Figure 2 illustrates the working principle of the proposed biosensing platform. After molecular assembly (a), The polymers are prepared via RAFT-polymerization (b) to ensure uniformity in the molecular weight, followed by removing the template from the polymer via dialysis (c). Once the templates are fully removed, the synthesized target receptors are attached onto the gold electrode, forming a brush-like monolayer assembly (d). During analyte detection, when the target binds to the molecular receptor, the conformation of the polymer changes which can be measured electrochemically using the redox tags (vinyl ferrocene) (e). The selective target rebinding is based on the weak interaction between the functional monomers in the polymer and 4-NP such as hydrogen bonding and the π-πstacking force (f).
The immobilization of polymer on gold surface is confirmed using electrochemical impedance spectroscopy (EIS). As shown in Figure 3, the impedance is gradually increased with the incubation time, indicating successful attachment of the polymer on the gold surface. Figure 4 shows the process of determining the binding affinity of the templated polymers using gold nanoparticles (AuNPs) and UV-vis. Figure 5 (a) indicates that the MIP exhibits higher affinity toward 4-NP compared with the NIP. In Figure 5 (b), the selectivitymolecular templating is demonstrated by comparing wtih 3-nitrophenol (3-NP). In Figure 6, it is shown that, as the concentration increased, the charge transfer is promoted in the MIP due to the collapsing of the polymer upon target recognition. NIP, on the other hand, shows negligible response to the 4-NP, indicating no significant conformational change for the polymers. Figure 7 (a) summarizes the calibration curves for both MIP and NIP. The templated polymer-based sensor shows a linear response to 4-NP solution, indicating that the proposed target receptor and the sensing platform is capable of detecting 4-NP. Figure 7 (b) shows the calibration curves when the templated polymer-based sensor is exposed to either 4-NP or 3-NP, suggesting a promising specificity towards 4-NP. In summary, an electrochemical sensing platform has been successfully developed, and the selectivity of the 4-NP imprinted polymers has been confirmed. The results demonstrate that 4-NP imprinted polymer undergoes a conformation change during specific target recognition. We envision that the proposed sensing platform can potentially be used in other applications such as neurotransmitter detection and drug delivery monitoring.
References:

[1] A.P.F. Turner, Chem. Soc. Rev., 2013, 42, pp 3184-3196.
[2] K. Haupt and K. Mosbach, Chemical reviews, 2000, 100(7), pp 2495-2504.

Figure 1