Accepted Manuscript

Title: Molecularly imprinted polymer-based core-shells (solidVs hollow) @ pencil graphite electrode for electrochemicalsensing of certain anti-HIV drugs

Author: Bhim Bali Prasad Kislay Singh

PII: S0925-4005(16)32080-9DOI: http://dx.doi.org/doi:10.1016/j.snb.2016.12.109Reference: SNB 21483

To appear in: Sensors and Actuators B

Received date: 7-10-2016Revised date: 21-11-2016Accepted date: 21-12-2016

Please cite this article as: Bhim Bali Prasad, Kislay Singh, Molecularly imprintedpolymer-based core-shells (solid Vs hollow) @ pencil graphite electrode forelectrochemical sensing of certain anti-HIV drugs, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2016.12.109

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Molecularly imprinted polymer-based core-shells (solid Vs hollow) @ pencil graphite

electrode for electrochemical sensing of certain anti-HIV drugs

Bhim Bali Prasad*1 and Kislay Singh1

Analytical Division, Department of Chemistry, Institute of Science, Banaras Hindu University,

Varanasi-221005, India

*Corresponding author: Prof. B. B. Prasad, E-mail address: prof.bbpd@yahoo.com, Phone +91 9451954449; Fax:

+91 542 22368127; K. Singh, E-mail address: kislaysingh1719@gmail.com

1 contributed equally

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Graphical abstract

Justification

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, 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, to gain better electroconductivity on account of rapid diffusion

of test analyte across the inner and outer surfaces in cooperation with the molecular

exchange between analyte molecules.

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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.

Keywords: solid/hollow core-shells, core-shells modified imprinted electrochemical sensors,

lamivudine, zidovudine, ferricyanide probe, differential pulse anodic stripping voltammetry

1. 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-

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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

[3-7]. 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 [18-21]. 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

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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

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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]:

𝜕µ𝑐

𝜕𝑡= 𝐷0

𝜕2µ𝑐

𝜕𝑟2 0 < r < Rc (1)

𝜕µ𝑠

𝜕𝑡= 𝐷𝑠ℎ

𝜕2µ𝑠

𝜕𝑟2 Rc < r < Rp (2)

where µc (= rcc) and µs (= rcs) are chemical potentials of diffusion species within the core

(radius RC) and shell ( radius RP), cc and cs are the concentrations in the inner core and outer

shells, and Do and Dsh 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

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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).

2. Experimental

2.1. Chemicals and Reagents

Demineralized triple distilled water (conducting range 0.06–0.07 × 10−6 S cm−1) was used

throughout this work. Melamine (mel), acryloyl chloride (AC), potassium ferricyanide, dimethyl

formamide (DMF), methanol, agarose, and hydrofluoric acid (HF, 40% v/v) were purchased

from Loba chemie (Mumbai, India). Dimethylsulphoxide (DMSO), ethanol, and methanol were

purchased from Spectrochem Pvt. Ltd. (Mumbai, India). Ethylene glycol dimethacrylate

(EGDMA), α,α’-azoisobutyronitrile (AIBN), tetraethoxysilane (TEOS, 98%), ammonium

hydroxide solution (31.5% NH3), and γ- methacryloxypropyltrimethoxy silane (γ-MPS) were

purchased from Aldrich company. 3TC, AZT, and acridine orange were obtained from Sigma–

Aldrich (Steinheim, Germany). All interferents were purchased from Fluka (Steinheim,

Germany). The supporting electrolyte used was moderately basic phosphate buffer solution (pH

7.4, ionic strength 0.01 M), since both AZT and 3TC are prone to destabilization owing to

hydrolysis, oxidation and photolysis in acidic and basic medium [31]. The stock solution of 3TC

(500 µg mL−1) was prepared in water and stored in a dark glass bottle below - 4 oC, in a

refrigerator for a week. For the preparation of stock solution of AZT (500 µg mL−1), 12.5 mg

AZT was dissolved in 2.5 mL NaOH ( 2.0 M ), 2.5 mL ethanol and 20.0 mL water. This was also

stored in dark but at the room temperature, 25 oC [7]. Standard stock solution of potassium

ferricyanide (0.10 mM) was prepared in water. All working solutions were prepared daily by

diluting respective stock solution with water. Human blood serum was obtained from the

Institute of Medical Science, Banaras Hindu University (Varanasi, India) and kept in a

refrigerator below - 4 oC, before use. Pharmaceutical samples, Nexvir S (claim: 150 mg

Lamivudine per tablet) and Retrovir (claim: 300 mg Zidovudine per tablet), were procured from

Nexus (India) and ViiV Healthcare UK Ltd., respectively.

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Pencil rods (2B), 2.0 mm in diameter and 5.0 cm in length, were purchased from HiPar,

Camlin Ltd. (Mumbai, India). The pencil graphite electrode (PGE) was used in this work

because of its larger electrochemical active surface area, higher electrochemical activity, good

mechanical stability, low cost, low background current and wide potential window. Descriptions

about instruments used in this work are provided in the supporting information Section S.2.

2.2. Synthesis of Functional Monomer

The monomer, TAT, was synthesized as reported elsewhere [32] (For details, vide

Supporting Information Section S.3).

2.3. Preparation of Vinyl Groups modified Silica Nanospheres (v-SiO2)

The preparation of v-SiO2 was carried out following a known recipe [25] (For details, vide

Supporting Information Section S.4).

2.4. Immobilization of MIP on the v-SiO2

Targets, 3TC or AZT (0.1 mmol) and TAT (0.2 mmol), were dissolved together in DMSO

(0.5 mL), followed by adding 0.058 g as-prepared v-SiO2. The mixture was sonicated to disperse

the v-SiO2 and to facilitate the formation of the complex between target and TAT. Subsequently,

EGDMA (0.5 mmol) and AIBN (0.003 g) were added to the above mixture followed by N2

purging. This pre-polymerization mixture was subjected to the free radical polymerization for 3 h

at 60 oC. The resulting polymer was collected by centrifugation. The target molecules were

extracted from the respective polymer adducts using 0.1 M HCl extractant for 30 min. The

extraction was continued till no template molecules were detected in terms of decrease of the

initial DPASV signal ascribed to ferricyanide probe. The obtained MIP is now template-free.

The corresponding non-imprinted core-shells (Cs-NIP) were also prepared following the above

procedure, but in the absence of template concerned.

2.5. Removal of v-SiO2

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The SCs-MIP and SCs-NIP could be converted to HCs-MIP and HCs-NIP simply by the

removal of v-SiO2 from the solid core with HF (40 %, v/v), for an hour treatment under dynamic

condition [24]. It may be noted that the aqueous HF has routinely been used for the etching of

silica, without any apprehension of hydrolysis of EGDMA in the polymer synthesis [33,34]. This

is because of the fact that the water molecules remain intact in acidic condition, without

producing hydrolyzing components (H+, OH-), as a consequence of the common ion effect in the

autoprotolysis equilibrium (H2O + H2O H3O+ + OH-).

2.6. Immobilization of SCs-MIP/HCs-MIP on the Surface of PGE

First core-shells (20.0 mg) were dispersed in 1.0 mL methanol and ultrasonicated for 20 min.

This suspension (15.0 µL) was spin coated on the PGE surface at 2500 rpm for 30 s. After

evaporation of methanol, the surface was protected with hot 5 µL agarose (2 %, w/v) by spin

coating and then dried at 30 oC for 1 h.

2.7. Voltammetric Procedure

For cyclic voltammetry (CV) and differential pulse anodic stripping voltammetry (DPASV),

core-shells modified (SCs-MIP/HCs-MIP) PGE was immersed into a cell containing 10.0 mL of

0.01 M phosphate buffer (pH 7.4) in the presence of potassium ferricyanide (0.10 mM, 50.0 µL).

Before CV and DPASV runs, the probe molecules were accumulated in the form of an electrical

double layer consisting an array of K+ and [Fe(CN)6]3− at − 0.5 V for 180 s. CV runs of

[Fe(CN)6]4− (reduced form at − 0.5 V) were scanned within the potential window − 0.3 to + 0.3

V at a scan rate 20 mVs-1 in anodic stripping mode. DPASV runs were recorded applying

modulation amplitude (25 mV), pulse time (50 ms) and step potential (5 mV) at a scan rate of 10

mV s−1 from − 0.3 to + 0.3 V to obtain initial run. This electrode was taken out from the cell

containing only phosphate buffer solution (pH 7.4, 10.0 mL) and then subjected to template

rebinding, under open circuit, for 10 min. This was again brought to the cell to measure the

difference in ferrocyanide oxidation current (I). Since dissolved oxygen present in the cell did

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not affect the current response, any deaeration of the cell content was not necessary. The limit of

detection (LOD) was calculated as three times the standard deviation for the blank measurement

in the absence of target analyte divided by the slope of the calibration plot. All experiments were

performed at 25 ± 1 oC.

3. Results and discussion

3.1. Polymer Characteristics

DPASV runs, as depicted in Fig. 1, correspond to the current response of standard probe

solution (0.1 M, 25.0 L), simply added in 10.0 mL phosphate buffer solution (pH 7.4), using

different type of PGE sensors modified with HCs-MIP, SCs-MIP, planar-MIP, and HCs-NIP

materials. SCs-MIP did not respond well owing to the smaller diffusion coefficient (D) of the

probe as a consequence of less porous and insulating nature of v-SiO2 (Fig. 1, curve b) (DSCs-MIP

= 3.15 x 10-6 cm2 s-1, DHCs-MIP = 4.60 x 10-6 cm2 s-1, and Dplanar-MIP = 1.54 x 10-6 cm2 s-1; as

calculated on the basis of Randles Sevcik equation) [35]. However, SCs-MIP was turned to be

more porous, when v-SiO2 was etched out with HF to obtain a hollow core. Consequently, HCs-

MIP responded the maximum development of anodic stripping current under the oxidation

process [Fe(CN)6]4- [ Fe(CN)6]

3-, of accumulated reduced species as ferrocyanide ions at -0.5

V (Fig. 1, curve a). As a matter of fact, HCs-MIP modified PGE showed approximately 1.4-fold

enhancement in anodic stripping current height, as compared to SCs-MIP-modified PGE. It

means that the hollow structure had a positive impact to improve the current, on account of the

typical diffusive flux (sh) and anticipated molecular exchange through bridges at the contact

point between proximate hollow-core-porous shells (see Introduction). Eventually, all diffusing

probe molecules traverse from vertically aligned HCs-MIP arrays to their contact points with the

electrode surface. As a proof of the concept that HCs-MIPs have relatively high conductivity (),

we have measured and compared the electrical properties, such as resistance and conductivity of

HCs-MIP, SCs-MIP, planar-MIP, and CNTs, all in pelletized form, using the two probe method

[36]. The corresponding results are shown in Table S1. Accordingly, HCs-MIP behaved better

than SCs-MIP and planar-MIP, and even superior than MWCNTs, in terms of conductivity. This

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was further confirmed by electrochemical impedance spectroscopy (EIS), wherein the Nyquist

plots (Fig. S1A) revealed relatively low charge (electron) transfer resistance (RCT (HCs-MIP) = 383.2

RCT (SCs-MIP) = 3182 RCT (planar-MIP) = 4450 and RCT (CNT) = 909.6 and thereby the

maximum electroconductivity to respond higher CV current (Fig. S1B) for probe (0.10 mM

[Fe(CN)6]3-/4- in 0.10 M, 5.0 mL KCl ) at HCs-MIP@PGE. The involved heterogeneous electron

transfer rate constant (ket = 1.87 x 10-4 cm s-1) for [Fe(CN)6]3-/4- redox couple on HCs-MIP@PGE

was found to be higher than those realized with other electrodes (For details, on two-probe

method and EIS measurements, vide Supporting Information Section S.5). In the case of SCs-

MIP, aforesaid diffusion formulations, however, turned somewhat ineffective to behave as

electro-catalytic CNTs. Thus, the anticipated diffusion along with the proposed molecular

exchange could be restricted within the solid core-shells, resulting in a diminished response (Fig.

1, curve b) for probe molecules. It is worth to note that the longitudinal diffusional flux

(across the monolith planar-MIP film on the electrode surface may involve relatively very

slow binding kinetics to respond much lower current (Fig. 1, curve c) compared to core-shells.

This aspect is already explained elsewhere [25,37]. Accordingly, compared with the MIP

prepared by traditional method or MIP microspheres, the HCs-MIP showed a relatively fast

binding kinetics as suggested on the basis of Langmuir and Scatchard data (For details, vide

Supporting Information Section S.6 and Table S2). This difference could be attributed to most of

the imprinted cavities, situated at the surface and proximity of outer/inner shell surfaces, with

apparently very high surface-to-volume ratio, enable them to be largely accessible for the

template molecules. The phenomenal imprinting effect in HCs-MIP was reflected from the fact

that the corresponding HCs-NIP was not competent to show specific binding of the analytes (Fig.

1, curve d).

FT-IR (KBr) spectra (Fig. S2) of templates (3TC and AZT), functional monomer (TAT),

MIP-template adduct, and MIP were compared with each other to propose a tentative binding

mechanism between monomer and template (Fig. S2 inset). The complexation between the

monomer and template(s) via hydrogen bonding was indicated by the downward shifts of their

respective key bands participating in the adduct formation (for details, vide Supporting

Information Section S.7).

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3.2. Surface Characterization

SEM and TEM studies were carried out to get an insight into the surface morphologies of

core-shells MIP. Additional EDS study was performed to explore elemental mapping and to

substantiate complete template retrieval, followed by total etching of v-SiO2 from SCs-MIP

adduct to obtain HCs-MIP. Surface morphologies of HCs-MIP adduct and HCs-MIP were

further studied using AFM three dimensional images. This revealed the thickness (83.72 nm) of

MIP layer almost same as was observed with SEM (side view) image (for details, vide

Supporting Information Section S.8, Fig. S3 A-G, and Fig. S4 a-d). The confocal microscopy

images (Fig. S5) of HCs-MIP (empty) and HCs-MIP (duly filled with the contrast material,

acridine orange) confirmed the existence of hole (hollow structure) within a core-shell.

3.3. Electrochemical Study

CV of potassium ferricyanide probe at bare PGE (without any MIP coating) showed

reversible (prone to quasi-reversible) oxidation and reduction peaks [peak separation, ∆Ep ( Epc-

Epa) = 150 mV] of the redox system, [Fe(CN)6]3−/[Fe(CN)6]

4−, in the phosphate buffer (pH 7.4).

The deviance from ideal reversible behavior of probe and the tendency to assume quasi-

reversibility could be accorded to the difficulty in oxidative stripping of reduced ferricyanide

from the electrical double layer formed at Eacc = - 0.5 V . However, upon modification of PGE

with HCs-MIP, SCs-MIP, and traditional MIP monolith film created a barrier and consequently

the quasi-reversible redox CV peaks (∆Ep = 120 mV) of probe was relatively decreased to a

certain extent (Fig. S6). The apparent surface coverage (= 20.0 %) by the polymer and BET

surface area of HCs-MIP/PGE were also evaluated and compared with SCs-MIP/PGE. The poor

surface coverage reflects a relatively thin coating of MIP with higher porosity on both exposed

concave and convex surfaces of HCs, despite having SBET (282 m2 g-1) just twice to that (142 m2

g-1) of SCs. (For details, vide Supporting Information Section S.9).

Various conditions for polymerization were optimized, such as polymerization time (3 h),

template extraction time (30 min), template-monomer ratio (1:2), and monomer-crosslinker ratio

(1:5) (For details, vide Supporting Information Section S.10 and Fig. S7). Applying these

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conditions, the maximum development of DPASV diminishing current (∆I) for ferricyanide

probe was achieved which is in commensurate with the amount of drug analytes added in the

voltammetric cell. The operating analytical conditions were also optimized for the maximum

development of ∆I of ferricyanide probe. Accordingly, the optimized parameters such as

accumulation potential (Eacc = - 0.5 V), the accumulation time (tacc = 180 s), and pH (7.4) of the

phosphate buffer [preferred medium of supporting electrolyte should be slightly basic (pH 7.4)

like blood to obviate the probable ionization of drugs [38]] were utilized for the analysis (For

details vide Supporting Information Section S.10 and Fig. S8). Herein, both K+ and [Fe(CN)6]3−

ions occupy and fill up the core, and then diffused to shell cavities governed by two coupled

Fick’s diffusion equations (Eqs. 1 and 2) in open circuit. However, these ions, being small ones,

could not inhabit the shell imprinted sites of test analyte, but rather again effectively diffused

toward electrode surface, under the influence of applied potential and accumulated there as an

electrical double layer [an array of K+ and [Fe(CN)6]3−], under the pool of electrostatic

interactions at - 0.5 V. At this potential, [Fe(CN)6]3− is first reduced to [Fe(CN)6]

4−and then after

anodically oxidized as [Fe(CN)6]3− under stripping mode to respond DPASV signal. For

quantitative analysis of 3TC (or AZT), the HCs-MIP modified PGE is always subjected to this

process at fore hand to record the initial signal of ferricyanide probe. There were some frivolous

initial current variations within ± 0.64 A which may be due to the matrix effect. However, this

would not affect the final result as we measure the relative change in current, upon addition of

the test analyte. The electrode is given water-washing treatment so as to remove extraneous

probe molecules from the core; and the shell cavities. Finally, this electrode is exposed to test

analyte solution (maintained at optimized pH 7.4) for 10 min, in an open circuit, manifesting

chemisorptions of template molecules. This electrode is again brought into the cell (containing

0.01 M phosphate buffer supporting electrolyte, pH 7.4) and added 50.0 L of 0.10 mM

ferricyanide probe for recording corresponding anodic stripping current in the similar manner as

stated above. This revealed a diminished current owing to the apparent constraint (blockage)

toward the passage of probe molecules by the accumulated test analyte in the MIP shell cavities.

With the increase of analyte concentration, current response for [Fe(CN)6]3−/4− redox couple was

observed to be further decreased. As the inter-conversion of [Fe(CN)6]3− / [Fe(CN)6]

4− redox

reaction occurs as a surface controlled process, the increase of target concentration decreases the

P a g e | 14

availability of pores at the electrode surface. Herein, the effective surface area is reduced due to

blocking of imprinted sites with the hydrogen bonded 3TC (or AZT), which consequently led to

the decrease in the DPASV response (Fig. 2A-B and Fig. S9A-D) and CV response (Fig. S10 A-

F) of probe. DPASV runs were found to be symmetrical in all sample matrices studied. On the

other hand, corresponding CV runs in real samples are somewhat drawn out (Fig. S10,C-F),

despite being electrodics involved to be a diffusion-controlled and pseudo reversible process, in

accordance with Randles Sevcik equation (ipa/ ipc 1, ipa vs plots with R2 = 0.968, figure not

shown Furthermore, voltammetric quasi-reversibility (∆Ep = 100-150 mV) is almost maintained

without any significant effect from complicated matrices of dilute real samples.For sake of

brevity, the effect of scan rate on CV at HCs-MIP/PGE is exclusively shown for aqueous

solution of probe (0.10 mM, 50 L) in phosphate buffer with both 3TC and AZT imprinted

cavities (Fig. S11 A,B). Accordingly, corresponding ipa vs plot is not found to be perfectly

linear (R2 = 0.973) (Fig. S11A,B Inset). This suggests a quasi-reversible characteristic of

electrode process as observed with real samples. This means effect of matrix is not pertinent in

the present instance. For the analyte quantification, we have preferred DPASV to CV owing to

its better sensing ability responding symmetrical signals in the sufficient time scale of

voltammetric measurements. Thus, the ∆I could be indirectly related to the concentration (C) of

test analyte in accordance with the linear regression equations (Eqs. 3 and 4); analyte recoveries

are calculated as (concentration determined/concentration taken) x100

•For aqueous solution:

3TC: ∆I (µA) = (0.022 ± 0.029) + (0.102 ± 0.001) C, (3)

n = 9, R2 = 0.9999

(Concentration range = 7.26 - 80.16 ng mL−1, LOD (3σ) = 2.23 ± 0.02 ng mL−1, recovery = 97-

104%)

AZT: ∆I (µA) = (0.004 ± 0.004) + (0.063 ± 0.001) C, (4)

n = 14, R2 = 0.9999

(Concentration range = 4.76 - 128.76 ng mL−1, LOD (3σ) = 1.26 ± 0.04 ng mL−1, recovery = 99-

102%)

As is evident from Fig. 2C-D, the diminished DPASV current (∆I) is sharply increased

with increasing concentration of electroinactive targets (3TC and AZT) which eventually became

P a g e | 15

constant due to binding sites saturation above 80.16 and 128.76 ng mL−1, respectively. The sharp

increase of ∆I, with distinctive slopes (0.102 ± 0.001 for 3TC and 0.063 ± 0.001 for AZT), upon

addition of test analytes could be attributed to their strong and higher binding affinities (KD = 104

order) in shell cavities and better electrode kinetics with probe (ket = 1.87 x 10-4 cm s-1). Both

analytes observed linear Langmuir and Scatchard plots. Accordingly, Table S2 depicts

comparative study of binding parameters (binding constant, KD and maximum diminished in

current, ∆Imax). This revealed higher KD and ∆Imax for AZT which suggested relatively strong

binding affinity and maximum number of binding sites accessible to AZT as compared to 3TC.

Furthermore, Langmuir and Scatchard KD and ∆Imax values obtained with HCs-MIP were more

favored to facilitate better binding affinity as compared to SCs-MIP and planar-MIP for both

analytes (For detail, vide Supporting Information Section S.6). Interestingly, HCs-NIP/PGE

showed some insignificant analyte adsorption which was completely washed away by water (Fig.

2A-B, curve k and p). This may be attributed to an excellent imprinting effect of both analytes, in

the present instance.

3.4. Interferences and Cross-reactivity

We have examined the electrochemical response of HCs-MIP and HCs-NIP-modified PGEs

with various interferents viz., dopamine (DA), glutamic acid (GA), cytosine (Cyt), thymidine

(Thy), cytarabine (Cytr), tyrosine (Tyr), phenylalanine (Phen), stavudine (D4T), and their

relevant mixtures as shown in Fig. S12. Accordingly, HCs-MIP /PGE was found to be slightly

responsive for the interferents when studied individually. In a parallel work with binary

(template-interferent 1:1, and 1:10) mixtures, the HCs-MIP modified electrode showed an

exclusive response for the template in the quantitative manner by means of stereo chemical

selectivity in terms of shape, size, and functional groups affinity. There is virtually no cross

reactivity between target and interferent(s) i.e, HCs-MIP imprinted with 3TC could not respond

AZT and vice-versa. Interestingly, the HCs-NIP-modified electrode revealed a very feeble

current response for interferents (Fig. S12), which could easily be washed away from the

electrode with water (0.5 mL, n = 2). As a safeguard against such non-specific adsorption, HCs-

MIP/PGE should also be given the similar washing treatment to avoid false-positives in the final

P a g e | 16

results. Although somewhat structurally identical interferents like D4T, Cytr, Thy and relatively

small molecules like Phen, Tyr, Cyt, GA, DA have a fair chance of approaching the imprinting

sites but still mismatch to the sites for binding. This reflects substrate-selective imprinting effect,

in the present instance. The substrate selectivity could also be attributed to the steric

conformations associated with different pyrimidine-based targets (3TC and AZT). Accordingly,

HCs-MIP imprinted for 3TC was selective for 3TC only and not responsive for AZT; and vice-

versa. The major factor which governed the selectivity of 3TC and AZT into their respective

molecular cavities was owing to the phenomenal imprinting effect. As a matter of fact,

imprinting factors (α = i HCs-MIP/i HCs-NIP) for both templates (3TC and AZT) were found to be as

high as 18.08 and 24.06, respectively using HCs-MIP/PGE (without water washings). The

selectivity coefficient (k) and relative selectivity coefficient (k’) for 3TC and AZT are

supplicated in Tables S3 and S4 (For details, vide Supporting Information Section S.11).

3.5. Stability and Reproducibility of the Proposed Sensor

To explore the precision of results using the proposed HCs-MIP/PGE sensor, multiple

DPASV runs were recorded for the analytes, 3TC and AZT (each 14.52 ng mL-1). The relative

standard deviation (RSD) in results was found within 0.33 %. Further to evaluate electrode-to-

electrode reproducibility, a series of as many as six modified electrodes were prepared in the

identical manner and tested for 14.52 ng mL-1 analyte. All electrodes responded quantitatively

(100%) with RSD 0.53 %. Regeneration of the modified electrode, after each DPASV

measurement, could be feasible employing the reported method of template retrieval, i.e., using

0.1 M HCl eluent under dynamic conditions. Current intensities of the analyte decreased to 5.12

% of the initial value, after being used for more than 55 rebinding–extraction cycles. Insofar as

exposure to the extractant (0.1 M HCl for both the analytes) for recycling of HCs-MIP is

concerned, the proposed sensor for both analytes was found to be chemically stable at the

working pH (7.4) and temperature (25 oC), without showing any deviance in DPASV response

up to 55 regeneration cycles. The stability of the proposed sensor was also examined by

intermittent recording of DPASV response of the standard analyte solution, on every third day,

over a period of one month. A similar conclusion could be withdrawn when the reproducibility

P a g e | 17

and ruggedness of the HCs-MIP electrode were examined in real environments. This

demonstrated that the prepared electrochemical sensor had excellent regeneration and

ruggedness, claiming a novel class of HCs-MIP electrodes for 3TC (and AZT) sensing at the

ultratrace level.

3.6. Analytical Validation

Under optimized operating DPASV conditions, the proposed sensor was also validated for the

evaluations of 3TC (and AZT) in human blood serum and pharmaceutical samples. The

corresponding results are depicted as following linear calibration equations between peak current

(∆Ip, µA) and concentration (C, ng mL−1), along with respective LODs and % recoveries.

•In human blood serum:

3TC: ∆I (µA) = (0.022 ± 0.016) + (0.101 ± 0.003) C, n = 7, R2 = 0.9989 (5)

(Concentration range = 8.92–75.92 ng mL−1, LOD (3σ) = 2.45 ± 0.01 ng mL−1, recovery = 99–

102%).

AZT: ∆I (µA) = (0.003 ± 0.008) + (0.061 ± 0.001) C, n = 6, R2 = 0.9984 (6)

(Concentration range = 6.82-115.99 ng mL−1, LOD (3σ) = 1.88 ± 0.03 ng mL−1, recovery = 99–

101%).

•In pharmaceutics:

3TC:

∆I (µA) = (0.004 ± 0.039) + (0.101 ± 0.001) C, n = 9, R2 = 0.9988 (7)

(Concentration range = 7.92–77.96 ng mL−1, LOD (3σ) = 2.52 ± 0.02 ng mL−1, recovery = 98–

102%).

AZT:

∆I (µA) = (0.006 ± 0.015) + (0.054 ± 0.002) C, n = 8, R2 = 0.9986 (8)

(Concentration range = 5.82-127.88 ng mL−1, LOD (3σ) = 1.77 ± 0.04 ng mL−1 Recovery = 99-

101%)

Notably, pharmaceutical samples for 3TC and AZT were diluted as many as 18940 and

51550 folds, respectively so as to move the detection within the range of detection limits and

also to mitigate the matrix effect to the larger extent. Any pretreatment such as deproteinization

P a g e | 18

and/or ultra-filtration of blood serum sample had deliberately been avoided in this work as this

may lead inaccuracies in the final results. Instead, the dilution of blood (1000-fold) was found to

be quite effective against matrix effect, and the sample behavior was almost approximated to that

of the aqueous solution. As a matter of fact, the slopes of calibration equations of all the real

samples studied were found to be close (with RSD 0.69 % for 3TC and 7.92 % for AZT) to that

of aqueous sample. Therefore, detection sensitivities realized with real samples could be

considered reliable and useful for clinical studies, particularly in controlling oral

supplementation of drugs to the HIV patients. The proposed sensor is validated comparing with a

known method [3,7] by means of student’s t-test [3TC: tcal (2.35) < ttab(3.18), AZT: tcal (2.92) <

ttab(4.30)]. It is also worth to compare the proposed MIPs sensor with other known methods for

3TC and AZT determinations (Table S5). Accordingly, the detection senstivity, i.e., LOD and the

practical range of quantification by most of the earlier electrodes were inferior to our sensor and

moreover, majority of them were not validated with real samples.

4. Conclusion

We have demonstrated, for the first time, an efficient hollow core-shell structural MIP based

PGE sensor for ultra-trace sensing of two anti-HIV drugs (3TC and AZT) in real samples,

without any cross reactivity and false-positives. We have compared the proposed sensor with

SCs-MIP/PGE, in terms of electrodics involved with the help of a ferricyanide probe. Diffusion

coefficient of probe molecules on HCs-MIP/PGE was found approximately 1.5 times more than

that realized with SCs-MIP/PGE. This could be attributed to the difference of chemical potential

of the diffusing species and the difference of equilibrium concentration of analyte between the

inner concave and outer convex surfaces of HCs-MIP. Therefore, HCs-MIP had a typical

behavior, better than CNTs, to gain better electroconductivity (atleast 1.3 fold higher current

response than SCs-MIP). HCs-MIP involved relatively fast ingress and egress of both analytes

exhibiting imprinting factors as high as 18.08 and 24.06 and LODs as low as 2.23 and 1.26 ng

mL-1, for 3TC and AZT, respectively. The wide linear concentration range of test analytes

[blood: 3TC (8.92-75.92) and AZT (6.82-115.99) ng mL-1] with HCs-MIP/PGE demonstrates a

P a g e | 19

phenomenal improvement of our work in comparison to the earlier known methods (Table S5),

in terms of monitoring therapeutic drug doses requisite for the treatment of HIV-patients.

Acknowledgements

Authors thank University grant commission, New Delhi for a research fellowship to one

of us (K.S). Instrumental facilities procured from Banaras Hindu University are also greatly

acknowledged. We also thank Dr. V. Ganeshan of our Department for his generous help in

executing EIS experiments.

Supporting Information

Figures showing EIS, FT-IR, SEM, AFM, confocal microscopy, optimization of polymerization

conditions and analytical parameters, DPASV, CV, and interferents study, Tables for

conductivity, Langmuir and Scatchard data, selectivity coefficient/imprinting factor and

comparison of different electrodes.

P a g e | 20

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P a g e | 24

Bhim Bali Prasad is currently working as a professor of Analytical Chemistry in the Banaras

Hindu University (BHU), Varanasi, India. He has mentored 25 Ph.D. students and published 115

research papers in several reputed international and national Journals. He received his B.Sc.

degree in Chemistry in 1972 and M.Sc. degree in 1974 from BHU. He obtained his Ph.D. from

BHU. He is a recipient of several national and international awards for his research contributions

in Analytical Chemistry and nano-materials. His research interests include environmental

chemistry, chromatography, electroanalysis, and detection principle for chemical analysis, nano-

technology, and development of biomimetic nano sensors using molecularly imprinted polymers

for clinical, pharmaceutical and biological analysis.

Kislay Singh is currently pursuing Ph.D. at Banaras Hindu University (BHU) under the

supervision of Prof. Bhim Bali Prasad. She received her B.Sc. degree in 2011 and M.Sc. degree

in 2013 from BHU. She is recipient of UGC meritorious research fellowship. Her research

interest lies in the field of chemical sensors, molecularly imprinted polymers, and electro-

analytical chemistry.

P a g e | 25

Figure legend

Fig. 1. DPASV response of ferricyanide probe (0.10 mM, 25.0 L) on different types of

modified PGEs: curve ‘a’ on HCs-MIP@PGE showed an approximately 1.4 fold higher DPASV

current than curve ‘b’ on SCs-MIP@PGE, on account of the lowest time response for typical

diffusive flux (sh) in hollow core-shells possessing superior electrical properties (D, ket, RCT,)

; curve ‘c’ is the current obtained on planar-MIP film revealing much slower diffusion limited

response time, on account of the longitudinal diffusion flux () across the film, and curve ‘d’ is

the response on HCs-NIP@PGE showing inability for analyte binding in the absence of

imprinted cavities in the shell.

Fig. 2. (A) DPASV runs in aqueous medium showing a decreasing trend of oxidative stripping

current height of reduced ferricyanide probe (0.10 mM, 50.0 l) upon the rebinding of different

concentration of 3TC in the MIP cavities (from a to j): 0.0, 7.26, 9.98, 14.02, 20.01, 29.84,

37.11, 44.97, 72.96, 80.16 ng mL-1 (on HCs-MIP/PGE), and (k) 80.16 ng mL-1 (on HCs-

NIP/PGE). Curve ‘a’ represents an initial run of probe which successively decreased (b → j)

upon analyte rebinding. [Operating conditions: Eacc = - 0.5 V, tacc = 180 s, modulation amplitude

= 25 mV, pulse time = 50 ms, step potential = 5 mV, scan rate = 10 mVs-1 for probe; supporting

electrolyte 0.01 M phosphate buffer, pH = 7.4]

(B) DPASV runs in aqueous medium showing a decreasing trend of oxidative stripping current

height of reduced ferricyanide probe (0.10 mM, 50.0 l) upon the rebinding of different

concentration of AZT in the MIP cavities (from a to o): 0.0, 4.76, 15.68, 28.48, 38.09, 46.04,

57.35, 68.26, 73.03, 79.67, 87.32, 93.66, 101.69, 120.54, 128.78 ng mL-1 (on HCs-MIP/PGE),

and (p) 128.78 ng mL-1 (on HCs-NIP/PGE). Curve ‘a’ represents an initial run of probe which

successively decreased (b → o) upon analyte rebinding. [Operating conditions same as above]

(C) Calibration plot (I vs C) showing a sharp rise (slope = 0.102 ± 0.001) in probe current

owing to the higher binding affinity of 3TC, till the saturation of binding sites at 80.16 ng mL-1 is

attained.

(D) Calibration plot (I vs C) showing a sharp rise (slope = 0.063 ± 0.001) in probe current

owing to the higher binding affinity of AZT, till the saturation of binding sites at 128.78 ng mL-1

is attained.

P a g e | 26

[The sharp rise in probe current (I) in both Fig. 2C and 2D is due to instantaneous rebinding of

analyte molecules in molecular cavities which block the commensurate amount of probe

molecules to be diffused under electrostatic pool to the electrode surface. The consequent

decrease in current (I) is shown with error bar accounting standard deviation in the values with

the help of software (Microsoft Office Excel)].

Scheme 1. Schematic protocol of the preparation of SCs and HCs-MIP modified PGEs. The

inserted diagram sketches the internal structure of HCs-MIP and introduces notions used in the

equations: 1 and 2, Rp and Rc for the radii of the outer core and inner core, respectively, Dsh and

Do for the diffusivities in the mesoporous shell and in the core, respectively, and sh for the

diffusional flux in the shell.

P a g e | 27

Fig 1

Fig 2

P a g e | 28

Scheme 1