Molecularly imprinted polymer-based core-shells (solid vs hollow) @ pencil graphite electrode for electrochemical sensing of certain anti-HIV drugs

Highlights

• Fabrication of a new molecularly imprinted polymer (MIP) decorated core-shells (solid and hollow) as a sensing material for anti-HIV drugs, lamivudine and zidovudine.

• This analysis is performed in real samples, without any cross-reactivity and false-positives.

• Hollow core-shells MIP was found better than solid core-shells MIP in terms of typical behavior, akin to CNTs, HCs-MIPs undergo rapid diffusion of test analyte across the inner and outer surfaces in cooperation with the molecular exchange between analyte molecules.

Abstract

The present work describes a new, simple, and easy method for the fabrication of molecularly imprinted polymer-based core-shells (solid and hollow) @ pencil graphite electrode for sensing anti-HIV drugs, lamivudine and zidovudine, in real samples. For this, an imprinted polymer was developed on the surface of vinylated silica nanospheres to obtain modified solid as well as hollow core-shells. In this work, respective electrodics in terms of analyte diffusion for binding and electrode kinetics of both modified solid and hollow core-shells were compared using a ferricyanide probe with cyclic voltammetric and differential pulse anodic stripping voltammetric methods of transduction. Whereas modified solid core-shells evolved unilateral diffusion of probe/analyte molecules, the corresponding hollow core-shells were found to be relatively better owing to their bilateral diffusions into molecular cavities. Indirect detections of electroinactive targets chosen were feasible with the help of probe using imprinted hollow core shells modified electrode with limits of detection as low as 2.23 and 1.26 (aqueous sample), 2.45 and 1.88 (blood serum), and 2.52 and 1.77 ng mL⁻¹ (pharmaceutics) for lamivudine and zidovudine, respectively.

Introduction

Lamivudine [(−)-4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2(1H)-one], a negative enantiomer of a dideoxy analogue of cytidine, is commercially known as 3TC. Although 3TC has a very low cellular cytotoxicity, it can be absorbed initially in blood with 80% bioavailability. Notably, 3TC can be used for the treatment of chronic hepatitis B with lower dose than that required for HIV. On the other hand, zidovudine, 1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione, an analog of thymidine, called as ‘azidothymidine’ (AZT), is widely used in the treatment of HIV infection in patients with or without AIDS [1]. Since both 3TC and AZT are found intracellularly as 5-triphosphate metabolites, the combination of both drugs is normally used in HIV treatment. In view of the medicinal and pharmacological significances of 3TC and AZT [2], their regular monitoring to decide the level of oral supplementation is an important analytical agenda. This warrants the development of highly sensitive sensors. In this context, simple electrochemical techniques for sensing anti-HIV drugs have been attempted using mercury and carbon electrodes [3], [4], [5], [6], [7]. However, these were found to be incompetent to evaluate stringent limits of drugs with high specificity. In the present work, we have endeavored to fabricate a highly stable, sensitive, and selective electrochemical sensor to ensure safe administration of therapeutic drug doses of 3TC and AZT to HIV patients.

In order to induce the specificity of analysis in complicated matrices of real samples, we have relied upon the most burgeoning technique of molecular imprinting. This technology is capable of synthesizing various tailor-made synthetic materials called molecularly imprinted polymers (MIPs) that can specifically recognize targeted molecules [8]. Simply put, MIPs are synthetic receptors prepared with the signature of template molecules that serve as a mould for the formation of complementary binding sites [9]. The past few decades witnessed the extensive applications of MIPs in various fields of chemical analysis such as purification/separation [10], [11], chemo/biosensor [12], catalysis [13], [14], drug delivery [15], [16], and so on. Recently, two comprehensive reviews on the recent advances in molecular imprinting including versatile perspectives, challenges and applications were published [8], [17]. Notably, both drugs, 3TC and AZT, have been evaluated chromatographically using their respective MIPs [18], [19], [20], [21]. However, MIPs-based electrochemical analysis of 3TC and AZT is not yet attempted because of their electro-inactive nature.

Core-shell molecularly imprinted polymer (Cs-MIP) have aroused increasing interest owing to their easy accessibility and favorable mass transport [22], [23]. The hollow nanospheres with many unique properties such as, high surface-to-volume ratios, a continuous wall with a hollow interior, low specific gravity, etc., have been found to play a vital role in the wide range of applications [24]. Therefore, we endeavored for the first time to introduce a hollow structure to the MIP network, which may allow a bilateral mass diffusion of analyte or probe molecules from the outer and inner interfaces of MIP layer. This is certainly different than routine pathways of longitudinal diffusion across the flat layer of traditional MIP films. Although solid core-shells MIP (SCs-MIP) structures have been reported to improve accessibility for the imprint molecules, the rebinding sites confined within the exposed surface of shell may not allow the template or probe molecules to have effective diffusion [25]. On the other hand, the hollow core-shells MIP (HCs-MIP) can apparently allow the diffusion on both inner and outer exposed surfaces. This would augment the diffusion of template (or probe) spectacularly toward recognition sites. We have compared diffusion aspect of analyte adsorption on both solid and hollow core-shells in this work and found that the HCs-MIP was more advantageous to deliver high level of sensitivity of the measurement. The present work describes a simple procedure for the preparation of HCs-MIP, involving a trifunctional monomer (2,4,6-trisacrylamido-1,3,5-triazine, TAT) in the presence of 3TC or AZT as model templates (chemical structures of TAT, 3TC and AZT are shown in Scheme 1). Using vinyl-bearing silica nanospheres (v-SiO₂) as the seed (or core) and subsequent polymerization in the presence of template(s) would result in the formation of a solid core-shell-MIP adduct. After removal of silica seed with concentrated hydrofluoric acid, the HCs-MIP could be obtained. The so-produced HCs-MIP for respective targets is immobilized over the electrode. This represents a nano structured hollow core surrounded by a MIP layer essentially having the properties of a fully porous spherical particle [26].

Our interest in HCs-MIP for the fabrication of nanosensors lies from the fact that one may induce a high level tunability of controlling shell thickness with creation of mesopores for the encapsulation of K₃[Fe(CN)₆] probe molecules in open circuit. After washing the electrode with water, all non-specifically adsorbed probe molecules from core are washed away but occluded probe molecules are retained within the shell cavities. However, such entrapped probe molecules could not inhabit in the shell in potentiostatic condition, but rather get transported to the electrode surface to register the development of current signal, under potentiodynamic oxidative stripping mode. With the introduction of drug at this stage, core space is again filled with drug solution which may observe a typical diffusion behavior toward analyte adsorption in shell cavities. Accordingly, the diffusive transport of molecular species, particularly encapsulated in HCs-MIP, may evolve a translational molecular dynamics for diffusion of drugs within the core to specifically occupy their respective molecular cavities in shell. Consequently, probe molecules are now transmitted (diffused) toward the electrode surface to raise a diminished current response, under the blocking effect of analyte bound to MIP-shell cavities. In addition to this diffusion behavior toward analyte adsorption in core-shells, it may be hypothesized a typical molecular exchange between the molecular ensembles in HCs-MIP and in the medium surrounding the HCs-MIP (Scheme 1). Our motivation for using hollow core-shell geometry (Scheme 1 inset) can be understood by considering the diffusion processes that limit the time response of analyte adsorption that eventually affects the electrochemical sensing. One may assume the following two coupled Fick’s diffusion equations, with the initial and boundary conditions, viable for analyte adsorption within the structure of the hollow-core-porous shell spherical particle of defined radius, r [27]:

$$\frac{\partial {\mathrm{\mu }}_c}{\partial t}=D_0\frac{{\partial }^2{\mathrm{\mu }}_c}{\partial r^2}0<r<R_c$$

$$\frac{\partial {\mathrm{\mu }}_s}{\partial t}=D_{sh}\frac{{\partial }^2{\mathrm{\mu }}_s}{\partial r^2}{R}_c<r<R_p$$

where μ_c (= rc_c) and μ_s (= rc_s) are chemical potentials of diffusion species within the core (radius R_C) and shell (radius R_P), c_c and c_s are the concentrations in the inner core and outer shells, and D_o and D_sh are diffusion coefficients in core and shell, respectively. Surface resistances at the internal, between the inner core and mesoporous shell, and external, between the mesoporous shell and the bulk liquid, boundaries can be assumed negligible. The external diffusion of analyte from bulk liquid to MIP mesoporous shell can be governed by an independent Fick’s law of diffusion process. Similar diffusion path is adopted by probe molecules for their adsorption in core-shells, before being transported to the electrode surface for the indirect measurement of test analyte. We anticipate that the diffusion behavior may also be caused by the formation of the bridges at the contact point, between MIP coated hollow core-shells, allowing an efficient molecular exchange between them. This, in turn, may improve molecular diffusitivity in core-shells in open circuit, followed by responding better current signal in potentiodynamic condition, in comparison to the SCs-MIP. The analyte diffusion within the HCs-MIP is thermodynamically driven by the difference of the chemical potential (μ) of the diffusing species and the corresponding difference of equilibrium concentrations between the inner concave and outer convex surfaces. Notably, it is reported that the outer diffusion of core material is significantly faster than the inner diffusion of the shell phase, similar to that observed in the case of carbon nanotubes (CNTs) [28]. Therefore, HCs-MIP may behave as CNTs in terms of inducing better conductivity as compared to SCs-MIP. As a proof of concept, we have followed the Crank model [29] to support the aforesaid diffusion processes applicable for a spherical system. Accordingly, a planar system will have a much slower diffusion limited time response than the same polymer presented as a particulate microsphere [30] (For details, vide Supporting information Section S.1).