A molecularly imprinted whatman paper for clinical detection of propranolol

Journal Pre-proof A molecularly imprinted whatman paper for clinical detection of propranolol Yeliz Akbulut, Adem Zengin

PII:

S0925-4005(19)31475-3

DOI:

https://doi.org/10.1016/j.snb.2019.127276

Reference:

SNB 127276

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

30 May 2019

Revised Date:

7 October 2019

Accepted Date:

12 October 2019

Please cite this article as: Akbulut Y, Zengin A, A molecularly imprinted whatman paper for clinical detection of propranolol, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127276

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A molecularly imprinted whatman paper for clinical detection of propranolol

Yeliz Akbuluta and Adem Zenginb,*

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Department of Chemical Engineering, Faculty of Engineering, Inonu University, 44280 Malatya, Turkey b Department of Chemical Engineering, Faculty of Engineering, Van Yuzuncu Yil University, 65080 Van, Turkey

Corresponding author;

Adem Zengin, PhD

Tel: +90 432 225 1024. Fax: +90 432 225 1030

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E-Mail: [email protected]

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Department of Chemical Engineering, Van Yuzuncu Yil University, Van-TURKEY

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Graphical abstract

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Highlights

A novel molecularly imprinted whatman paper surface (MIP@WP)fabricated via surface initiated reversible addition fragmentation chain transfer (SI-RAFT) polymerization. MIP@WP was successfully applied to selective detection of propranolol for therapeutic drug monitoring (TDM).

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The limit of detection (LOD) for propranolol was found to be 0.3 µg/mL which could meet TDM for propranolol.

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ABSTRACT

Determination of drug concentration in body fluids is important issue for clinical studies to

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arrange treatment of patients. In the present study, we concentrated on the preparation of

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propranolol-imprinted poly (N-acryloyl-L-phenylalanine) film on a paper surface for spectrophotometric detection of propranolol in human plasma samples. The surface characterization of the imprinted surface was carried out by attenuated total reflectance-

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fourier transform spectroscopy, x-ray photoelectron spectroscopy, scanning electron microscopy and water contact angle measurements. Rebinding isotherms and kinetics were

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also investigated and maximum adsorption capacity of the imprinted paper surface was found

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to be 64.3 mg/g with high imprinting factor (4.20). Moreover, the results for selectivity and regeneration ability of the imprinted paper indicated that propranolol selectively interacted with the imprinted paper and had satisfactory reuse without changing its adsorption capacity. Under optimized conditions, the imprinted paper surface had a limit of detection of 0.3 µg/mL with lower intraday and interday precisions for determination of propranolol. The proposed method was successfully applied to determine propranolol in plasma samples where it showed

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recoveries ranging from 97.0%-99.5%. The method was also compared with traditional ELISA method and the results showed that the proposed method is sensitive and selective. It is believed that the prepared paper-based molecularly imprinted polymers can be good alternatives to traditional drug assays in clinical practice. Keywords: Whatman paper, molecularly imprinted polymers, propranolol, plasma sample.

1. Introduction

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From past to present, the aim of new drug development has been the introduction of drug therapies with high therapeutic effect, but without side and/or toxic effects [1]. It is known that both therapeutic and undesirable effects of drugs are very closely related to drug dose.

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For many drugs, standard dose administration based on observations and experiments is

generally not a problem [2]. On the other hand, accurate adjustment of dosage of drugs is of

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paramount importance in the treatment of heart diseases, bacterial infections, epilepsy,

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asthma, immunosuppressive conditions, or psychiatric diseases [3-5]. Measuring the levels of drug present in body fluids in order to obtain information about the plateau concentration of drugs reached during treatment and to check whether the drug concentration has reached an

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adequate level for efficient treatment or to prevent toxicity is generally called therapeutic drug monitoring (TDM) [6]. In addition to assessing the degree of therapeutic response or

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preventing possible toxic and side effects, TDM is also effectively used in clinical follow-up

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and has increasing importance among laboratories in terms of obtaining information about the suitability of drug therapy, drug interactions and adverse effects [7]. Today, the test density of laboratories performing TDM constitutes about 3-5% of clinical laboratory tests. However, it is expected that this ratio will increase to a much higher level due to the increase in the average age of people and the use of drugs which must be taken or monitored continuously. Currently, drug assays are performed in the clinical studies using some

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immunoassays that are commercially available such as enzyme-linked immunosorbent assay (ELISA) [8, 9], enzyme immunoassay (EMIT) [10], and fluorescence polarization immunoassay (FPIA) [11, 12]. Although these assays are specific, they have some disadvantages such as cross-reactivity with endogenous compounds, requiring skills and specific instruments, short shelf life and single use of corresponding kits, excessive washing steps and also high cost [13, 14]. As a result, there is an urgent need for sensitive, selective, as well as rapid, low cost, and effective method for detection of drugs in body fluids for clinical

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tests. Molecularly imprinted polymers (MIPs) are often named as enzyme mimics or synthetic

antibodies due to their high selectivity and binding affinity towards target molecules [15, 16].

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MIPs are easily synthesized via surface initiated controlled/living radical polymerization techniques such as atom transfer radical polymerization (SI-ATRP) [17] and reversible

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addition-fragmentation chain transfer (SI-RAFT) polymerization [18] on flat or particle

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surfaces in the presence of template molecule, functional monomer, porogen and initiator. After polymerization, the template molecules are easily removed from the polymer network with a suitable solvent to form cavities which are same in terms of size, shape and

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functionality as the template molecule. MIPs have been extensively used in sensors, catalysis, separation and enrichment of some biologically important molecules due to their versatile

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properties such as high stability, selective reactivity towards the target molecule comparable

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with antibodies, low cost and easy preparation protocol [19-21]. Nowadays, paper is usually used in clinical and analytical chemistry due to its unique properties include (i) paper is thin, lightweighted, easy manufacture, containing of 100% cellulose and also easy biodegradability; (ii) storage and transportation of paper are easy; (iii) high surface area to volume ratio; (iv) good adsorption properties, (v) compatibility with biological samples (blood, urine etc.); (vi) easy chemical modification for immobilization of

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proteins and antibodies [22, 23]. Furthermore, paper can be disposed easily by incineration [24]. Recently, cellulose paper substrates with specific functions were used for construction of smart biosensors and “lab on paper” for effective and low cost biosensing. [25-27]. Paper based chromatography has been already used to separate and detection of some (bio)molecules such as amino acids, proteins and antibodies [28, 29]. Litmus paper and urinalysis dipsticks are generally used as paper-based detection system [30]. Paper is also used as a supporting materials for qualitative and/or quantitative analysis for detection of food

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contaminants [31], environmental chemicals [32], small organic chemicals [33], pharmaceutical [32] and clinical biomarkers [34]. Recently, lateral-flow immonuassays [35]

and vertical flow immunoassays [36] are commonly used as paper-based diagnostic platform

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for selective detection of a wide range of (bio)molecules using labeled antibodies. Moreover, paper surfaces was also applied to molecularly imprinted technology and several paper-based

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molecularly imprinted membranes were prepared via UV-induced free radical polymerization

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to detect some important (bio)molecules in body fluids with paper spray ionization mass spectrometry [37-39]. However, the reported methods have some limitations in terms of poor sensitivity, ion suppression, and narrow dynamic ranges. However, as far as we know, there is

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no report about paper-based MIPs for monitoring concentrations of any drug coupled with a

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simple UV-vis spectrophotometer.

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Propranolol is a non-selective type of beta blocker used in the treatment of hypertension [40]. It is the first beta blocker to be developed successfully and remains the only drug that has proven effective in preventing migraine development in childhood [41] . Drug monitoring of propranolol is important and at high dosage of propranolol, it has negative side effects on the cardiovascular system that reduces cardiac contractility, sinus rate and intraventricular conduction [42]. Moreover, it can cause nervous toxicity that can lead to coma or convulsions 5

[43]. Thus, the monitoring of propranolol is vital to avoid the aforementioned side effects of the drug. Herein, we report an effective, selective, rapid and low-cost analytical method for detection of propranolol in human plasma samples by molecularly imprinted whatman paper (MIP@WP) coupled with UV-vis spectrophotometer. For this purpose, the propranolol-imprinted paper surface was synthesized in the presence of N-acryloyl-L-phenylalanine (NAPAL, monomer), methylenebisacrylamide (MBAAm, cross-linker), propranolol (template molecule),

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azobisisobutyronitrile (AIBN, initiator) and DMF/H2O (porogen). The prepared MIP@WP was characterized by a combination of several characterization techniques such as attenuated total reflectance- fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron

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spectroscopy (XPS), scanning electron microscopy (SEM) and water contact angle

measurements. The rebinding isotherms and kinetics were investigated in detail. The

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selectivity and regeneration ability of the MIP@WP was also studied. Additionally, the

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MIP@WP was also used as a selective sorbent for separation and detection of propranolol in plasma samples and the results of the proposed method were also compared with the ELISA

2. Experimental

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method.

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2.1. Materials and reagents

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All chemicals were purchased from Sigma-Aldrich (Germany) at highest purity and used without further purification unless otherwise noted. Azobisisobutyronitrile (AIBN, 98%) was purchased from Acros Organics and recrystallized from diethyl ether and stored at -20 °C until used. Dichloromethane (DCM), ethanol, methanol, and tetrahydrofuran (THF) were of HPLC grade. Human plasma samples were also provided by Sigma-Aldrich (Catalog No: P9523). Propranolol, atenolol, pindolol and N,N´-methylenebisacrylamide (MBAAm) were

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purchased from Alfa Aesar. N-acryloyl-L-phenylalanine (NAPAL) and chain transfer agent, 2-[(butylsulfanylcarbonylthiosulfanyl) propionic acid] (BCPA), were synthesized according to the literature [44, 45] . Ultrapure deionized water was used throughout the experiments 2.2. Apparatus Surface morphology of the paper surfaces was determined by JEOL JSM 6060 LV at accelerating voltage of 15 kV. The ATR-FTIR spectra were recorded by a Thermo Nicolet 6700 spectrometer. XPS analysis was carried out with Al Kα as an X-ray source. A Shimadzu

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UV-2550 spectrophotometer was used for UV-vis analysis. Water contact angle (WCA) of the prepared paper surfaces was determined by drop shape analyzer (DSA 100, Krüss). 5 µL of

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water was dropped on the surface and left for 5 s for equilibrium at room temperature.

2.3. Pretreatment of paper surfaces and covalent attachment of BCPA on paper surfaces

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A piece of Whatman No 1 filter paper (WP, 0.5 cm x 0.5 cm, 0.013 g) was first washed with

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plenty of acetone and ethanol and subsequently placed into an aqueous NaOH solution (8 wt %, 25 mL) and stirred on a shaker for 24 h at room temperature. The paper surface was repeatedly washed with ethanol until pH of the solution reached 7.0. Then, the paper sample

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was washed with DCM (5 x 20 mL) to remove ethanol in the paper sample and used without any drying for esterification reaction between –OH groups on the paper surface and –COOH

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group of BCPA [46].

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~ 13 mg of pretreated paper surface was put into 10 mL DCM solution of BCPA (0.364 mmol). N,N´-Dicyclohexylcarbodiimide (DCC; 0.4 mmol) was dissolved in 2 mL DCM and added to the solution at room temperature. 4-(Dimethylamino)pyridine (DMAP; 0.364 mmol) was dissolved in DCM (2 mL) and slowly added to the mixture [47]. The solution was shaken for 48 h at room temperature. After the reaction, the paper surface was successively washed with DCM, THF and methanol, respectively. The final yellowish paper was dried with a hair

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dryer and stored in a vacuum desiccator for further usage. The BCPA-coated paper surface is denoted as BCPA@WP in the rest of this paper.

2.4. Synthesis of MIP/NIP@WP 1.5 mmol NAPAL and 0.3 mmol propranolol were dissolved in 20 mL of DMF/H2O (3:1, v/v) and left to stand for 2h at +4 °C to form the pre-polymerization complex. A piece of BCPA@WP (~ 15 mg) was put into the pre-polymerization mixture. Subsequently, 7.4 mmol

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MBAAm and 0.005 mmol AIBN were added to the solution and the reaction vessel was sealed and the mixture was bubbled with nitrogen for 15 min on an ice bath. The

polymerization was carried out at 60 °C for 8h. After polymerization, MIP@WP was

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recovered from the solution and washed with DMF, methanol and H2O until the color of the supernatant was clear. Then, the imprinted propranolol was removed from the polymer

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network with 20 mL of methanol:acetic acid mixture (5:1, v/v) for 120 min. Lastly, the

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MIP@WP was rinsed with excessive methanol to remove residual acetic acid and dried with a hair dryer. As control, the non-imprinted Whatman paper (NIP@WP) was synthesized with

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aforementioned protocol in the absence of propranolol.

2.5. Rebinding studies of propranolol on the MIP/NIP@WP

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To examine the static adsorption properties, a piece of MIP/NIP@WP was put into a solution

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containing different amounts of propranolol (0.1-1.3 mg/mL) in 10 mL of phosphate buffer (PBS, 0.01M, pH 7.4) in separate erlenmeyers and stirred on an orbital shaker for 60 min at room temperature. After adsorption, the paper surface was recovered from solution and the final concentration of propranolol in the supernatant was determined by UV-vis spectrophotometer at 290 nm with the following equation: Q = (Ci – Cf)V/m

(1)

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where Qe (mg/g) is concentration of propranolol at equilibrium, Ci (mg/mL) is the initial concentration of propranolol, Cf (mg/mL) is the final concentration of propronolol after incubation, V (mL) is the total volume of the solution, and m (mg) is the mass of the paper surface.

10 mg of MIP/NIP@WP was put into propranolol solution (1.0mg/mL in 10 mL of PBS) and

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stirred on a shaker for different time intervals (5-90 min) at room temperature. After adsorption, the paper surface was recovered from solution and the final concentration of

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propranolol in the supernatant was determined by UV-vis spectrophotometer at 290 nm.

To test the selectivity of MIP/NIP@WP, atenolol and pindolol were chosen as structural

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analogues of propranolol. Then, 10 mg of MIP/NIP@WP was separately placed into

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propranolol, atenolol and pindolol solutions (1.0 mg/mL in 10 mL of PBS) and stirred on an shaker for 60 min at room temperature. After adsorption, the paper surfaces were recovered from the solutions and the final concentration of propranolol and its analogues in supernatant

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was determined by UV-vis spectrophotometer at 290 nm.

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2.6. Selective separation and determination of propranolol in human plasma sample

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To demonstrate the applicability of MIP@WP, propranolol-free plasma samples were selected as a clinical sample. First, to eliminate the interference effect as much as possible the plasma sample was pretreated as reported previously [48]. Briefly, 10 mL of the plasma sample was mixed with 10 mL of acetonitrile to induce protein precipitation. After filtration, the supernatant was spiked with propranolol with a final concentration ranging from 1.5 µg/mL to 12 µg/mL. Subsequently, 10 mg of MIP@WP was put inside the spiked samples and stirred

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for 60 min. After incubation, the MIP@WP was removed from the solution and the concentration of propranolol was determined by UV-vis spectrophotometer. Additionally, the concentration of propranolol in the spiked samples was determined by the ELISA method. 3. Results and discussion Growth of the molecularly imprinted polymers on different surfaces via SI-RAFT polymerization consists of two main steps: (1) Covalent attachment of RAFT agents onto surfaces and then, (2) Polymer layer growth in the presence of monomer, template, cross-

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linker and initiator [49, 50]. In this case, as illustrated in Scheme 1, after pretreatment of the paper surfaces, the RAFT agent BCPA, was covalently attached to the paper surfaces. After binding of BCPA, the paper surface was now ready for molecular imprinting via SI-RAFT

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polymerization in the presence of NAPAL (monomer), propranolol (template), MBAAm (cross-linker), AIBN (initiator) and DMF/H2O (porogen). After polymerization, specific

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cavities form which are complementary to propranolol in terms of shape, size and

suitable solution.

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3.1. Selection of monomer

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functionality after removal of the imprinted propranolol in the polymer network with a

The effectiveness of any MIPs in terms of high adsorption capacity and selectivity factors was

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largely depend on type of the functional monomers. The true selection of the functional

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monomers allows formation of a stable pre-polymerization complex and further create selective sites in the imprinting process. Methacrylic acid (MAA) [51, 52], acrylamide (AAm) [53, 54] and 4-vinylpyridine (4-VP) [55, 56] are widely used as functional monomers in molecular imprinting technology. Recently, amino acid moieties containing functional monomers have been used for preparation of MIPs for different applications[57-60]. In this case, different monomers were studied to determine the effect of monomer type on adsorption

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capacity (Q) and imprinting factor (α) of MIPs. As shown in Table 1, Q and α values for MAA, AAm were nearly same and a small increment was obtained for 4-VP. The increasing of Q and α can be attributed to the presence both of hydrogen bonding acceptor group and π-π interactions between template and 4-VP [61]. Moreover, an obvious increasing was obtained for NAPAL and gave better Q and α. It is well known that the imprinting effect is greatly enhanced by the presence both of hydrogen bonding acceptor and donor groups in a monomer and/or a template molecule [62]. The presence both of hydrogen bonding acceptor and donor

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groups in NAPAL monomer lead formation of multiple hydrogen bindings between NAPAL and propranolol. Moreover, π-π interactions that are weaker than hydrogen bindings between phenyl groups of NAPAL and propranolol may contribute to form more stable pre-

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polymerization complex [63]. As a result, formation of multiple hydrogen bonds and π-π

interactions between NAPAL and propranolol, poly(NAPAL)-imprinted paper surface had

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higher Q and α values.

3.2. Characterization of MIP/NIP@WP

The MIP@WP, as well as BCPA@WP and the pristine WP surfaces, were firstly

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characterized by ATR-FTIR and the transmittance spectra are shown in Fig. 1. For the pristine WP (Fig. 1a), the characteristic bands of cellulosic structures ( hydroxyl stretching absorption

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at 3340 cm-1; broad stretching vibration of –CH in –CH3 and –CH2 groups at 2897 cm-1, their

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bending vibrations at 1428, 1365 and 1317 cm-1) were observed [64]. The BCPA was immobilized on the paper surface through typical esterification reaction. As shown in Fig. 1b, the band at 1735 cm-1 could be attributed to C=O stretching vibration of BCPA indicating covalent attachment of BCPA to the paper surface. Note that, the other bands of cellulosic structure did not change due most probably to a very thin layer of BCPA on the paper surface and the thickness of this layer may be lower than the detection limit of ATR-FTIR [65]. After

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the growth of the MIP layer, the spectrum of the paper surface was almost changed (Fig. 1c). The bands located at 3062, 3031 and 1720 cm-1 could be attributed to –CH stretching vibrations of phenyl groups and C=O stretching vibrations of carboxylic acid groups of poly(NAPAL), respectively. In addition to these bands, the presence of amide I and amide II bands recorded at 1648 cm-1 and 1527 cm-1 also indicated that the MIP layer was successfully fabricated on the paper surface.

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XPS analysis was conducted to monitor the surface composition of the pristine WP, BCPA@WP and MIP@WP (Fig. 2). The surface chemical compositions estimated using the peak areas of the XPS spectra are listed in Supporting Information, Table S1. After the

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reaction of BCPA with hydroxyl groups on the paper surface, the carbon (C 1s) content was slightly increased while the oxygen (O 1s) content slightly decreased. In addition, a small

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amount of sulfur (0.95%) was detected with a binding energies at 226.0 eV ( S 2s) and 163.7

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eV (S 2p) indicating the formation of the BCPA layer on the paper surface. The narrow scan of S 2p for BCPA@WP (Fig. 2b inset) can be divided into two main components with binding energies at 163.5 eV and 162.5 eV, attributed to C-S and C=S, respectively. After the

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polymerization on BCPA@WP, the carbon content increased and the oxygen content decreased (Supporting Information, Table S1). As shown in Fig. 2c, the appearance of N 1s

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peak at about 400.0 eV also supported the formation of the MIP layer on the paper surface.

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Narrow scans of C 1s and N 1s for MIP@WP surface (Fig. 2d and 2e) showed the typical fingerprints of poly(NAPAL) with binding energies located at 288.4 eV ( O-C=O/N-C=O), 287.0 eV (C-O/C-S), 285.4 eV (C-N) and 285.0 eV (C-C/C-H) for C 1s and 400.0 eV (C-N) for N 1s. Moreover, after the polymerization, sulfur atoms located at the end of the polymer chains cannot be detected due to their low amounts (XPS method measures the atomic concentration greater than 0.1 %) [66].

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The morphology of the prepared paper surfaces was examined by SEM. SEM photomicrographs of the surfaces are presented in Fig. 3. There was no difference between pristine WP and BCPA@WP due to the presence of a very thin layer of BCPA on the paper surface (Fig. 3a and 3b). After the formation of the polymer network, the surface morphology changed and the coating of the polymer was clearly distinguished as seen in the high magnification SEM photomicrograph (Fig. 3c). In addition, the morphology of the NIP@WP

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surface (Fig. 3d) was similar compared with the MIP@WP surface due to the same preparation protocol except for not including the template molecule.

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The wettability of the prepared surfaces was investigated by water contact angle (WCA)

measurements and WCA images are shown in Fig. 4. Due to the hydrophilic nature of pristine

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WP, a low WCA of ~ 9.0° was observed. After the covalent attachment of the BCPA on the

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paper surface, the wettability of the surface was decreased and the WCA of the surface dramatically increased to ~ 112° due to butyl groups (R-group of the BCPA) which is located on the outermost of the paper surface. However, the WCA of the surface was drastically

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decreased to ~ 69.0° after polymerization. The transition from hydrophobic to hydrophilic character was caused by the formation of the relatively hydrophilic polymer network.

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Moreover, the NIP@WP surface was similar wettability compared with MIP@WP due to the

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same chemical composition.

3.3. Optimization of preparation conditions for MIP/NIP@WP To obtain the maximum adsorption capacity for propranolol on the MIP@WP surfaces, the effect of several synthesis parameters (amount of propranolol, polymerization time, type of washing solution and washing time for effective template removal) were studied in detail. Note that in order to increase of the measurement precision, the amount of propranolol and

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MBAAm was fixed at 1.5 mmol and 7.4 mmol, respectively, for all the optimization studies. In addition, initial propranolol concentration was 1.0 mg/mL and adsorption time was 60 min for equilibrium adsorption conditions throughout the optimized studies at 25 °C. The key parameter for preparation of MIPs with high recognition ability is formation of stable pre-polymerization complex. The stabilization of pre-polymerization is also directly depend on monomer-template interactions as well as template concentration. To understand the effect of concentration of propranolol in the preparation of MIP@WP, batch adsorption was carried

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out with different concentration of propranolol and the other synthesis conditions were kept constant. The binding capacities of MIP@WP increased with the increase in propranolol amount (Fig. 5a). However, there was an obvious decrease in adsorption capacity of

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propranolol when the amount of propranolol was higher than 0.3 mmol. It could be attributed to the presence of higher concentration of propranolol in the pre-polymerization mixture

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which lead to form large template aggregates and the pore diameter of cavities obtained after

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polymerization are an order of magnitude higher than the templates exists result in extremely poor imprinting effect [67]. So, it could be concluded that the optimum amount of propranolol

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for the specific adsorption capacity of MIP@WP was fixed at 0.3 mmol.

The polymerization time is the parameter to reach the maximum adsorption capacity and

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higher numbers of the imprinted sites for any molecularly imprinted polymer. To obtain

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higher adsorption capacity and imprinting effect, SI-RAFT polymerization time was adjusted from 0.5 h-12 h. As shown in Fig. 5b, the rebinding capacities of MIP@WP significantly enhanced by increasing the SI-RAFT polymerization time and reached maximum rebinding capacity at 8.0 h and then showed a decrease in rebinding capacity during further SI-RAFT polymerization time. This is most probably due to the fact that the thickness of the imprinted layer increased as the polymerization went on and the formation of too thick polymer layer

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also block the transport of propranolol to specific cavities on the paper surfaces. So, the rebinding process for propranolol becomes more difficult when as the polymerization time was longer than 8.0 h.

The extraction time is also a crucial parameter and as shown in Fig. 5c, with the increase in washing time, the rebinding capacities also increased which resulted from the formation of more and more specific recognition cavities. The maximum rebinding capacity was achieved

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for washing time of 120 min. On the contrary, extending the washing time caused the reduction of rebinding capacity of propranolol. It was assumed that further washing time

could partially degrade the imprinted cavities during the extraction process [68]. Removal of

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the template molecule is also notably important for the construction of specific binding cavities for molecularly imprinted polymers to achieve higher rebinding capacity and

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selectivity towards the template molecule. Herein, different extraction solvents or mixtures

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with different ratio of volumes were used for the removal of propranolol from the imprinted sites. As depicted in Fig. 5d, the maximum rebinding capacity was observed when a mixture of methanol and acetic acid (5:1, v/v) was used as an extraction solution for removal of

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propranolol from the MIP@WP surfaces. 3.4. Binding analysis of MIP/NIP@WP

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Rebinding properties of the fabricated MIP/NIP@WP were studied in detail by static

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adsorption experiments. The adsorption isotherms of MIP/NIP@WP were investigated and mathematically modeled to collect more information for the imprinting process. As depicted in Fig. 6a, the binding amount of propranolol on both MIP/NIP @WP increased with the increase in initial propranolol concentration and MIP@WP showed much higher adsorption capacity for propranolol than NIP@WP indicating the presence of the selective binding sites on the MIP@WP. Additionally, as the initial concentration of propranolol was kept at 1.0

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mg/mL, the adsorption capacity of MIP@WP reached 64.3 mg/g which was higher than that of NIP@WP (14.2 mg/g). These results also implied that the rebinding ability of propranolol was much higher on the MIP@WP than for NIP@WP. Moreover, the Scatchard adsorption model [69] was used to better understand the adsorption mechanism of MIP/NIP@WP. As indicated in Fig. 6b, the Scatchard plot of MIP@WP was linear indicating high binding affinity of the molecularly imprinted polymer and not surprisingly, NIP@WP displayed a non-linear plot indicating the non-specific adsorption of propranolol on NIP@WP. Moreover,

model were 73.1 mg/g and 0.337 mg/mL, respectively.

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the calculated theoretical Qmax and Kd for the MIP@WP surfaces with the Scatchard equation

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The rebinding kinetics in predetermined time intervals (5-90 min) were investigated to

determine the binding mechanism of the fabricated MIP/NIP@WP. As indicated in Fig. 7a,

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the rate of adsorption on the MIP@WP was fast within 45 min and reached equilibrium in a

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period of 60 min. It could be easily seen that NIP@WP showed relatively slower adsorption tendency and lower adsorption capacity than MIP@WP. The fast adsorption and high adsorption amount of MIP@WP most probably originated from the presence of abundant

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specific cavities on MIP@WP which enhance the mass transport. Moreover, the pseudo-firstorder and pseudo-second-order models [70] were applied to the kinetic results for MIP@WP

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to obtain more detailed information about the dynamic adsorption process. As shown in Fig.

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7b and 7c, the correlation coefficient (R2) of the pseudo-second-order model was higher than the other. This is interpreted as showing that the pseudo-second-order model had a better fit and the adsorption took place through chemical interactions between propranolol and MIP@WP surfaces [70]. The adsorption capacity of MIP@WP in this work higher than that of other reported studies and the equilibrium adsorption time of propranolol on MIP@WP is comparable or lower than that of other studies. Ma et al. used propranolol-imprinted polymer

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microspheres with surface epoxy groups and subsequently coupling was carried out with thiol-terminated poly(2-hydroxyethyl methacrylate to produce hydrophilic microspheres. The imprinted microspheres had an adsorption capacity of 35.0 mg/g with an equilibrium time of 90 min [71]. Zhang et al. prepared propranolol-imprinted monolithic column and found adsorption capacity as 17.93 mg/g with 16h equilibrium time [72]. Yoshimatsu et al. reported a propranolol-imprinted polymer based on composite nanofiber membrane showed an adsorption capacity of 19.1 mg/g with an equilibrium time of 60 min [73]. In another study,

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poly(MAA) beads were used for propranolol imprinting and reached 51.9 mg/g adsorption capacity within 2h [74]. Shen and Yen reported a new interfacial molecular imprinting

method based on Pickering emulsion polymerization using template-modified SiO2 colloidal

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particles for propranolol and the prepared MIP@SiO2 particles showed an adsorption capacity of 2.85-10.3 mg/g under different experimental conditions [75]. In our case, high adsorption

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capacity and fast adsorption kinetic could be attributed to not only the presence of thin

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polymer layer on the MIP@WP and also 3D compact fibrous structure of paper surface that can strengthen mass transportation.

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3.5. Selectivity and regeneration ability of MIP/NIP@WP The selectivity of MIP/NIP@WP was investigated and to approve the specific recognition

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ability of paper surfaces, two different analogues of propranolol were selected for comparison

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(Fig. 8a). As depicted in Fig. 8b, the MIP@WP showed higher adsorption capacity for propranolol. However, NIP@WP surfaces showed nearly same adsorption capacity for propranolol, atenolol and pindolol that much lower than MIP@WP surfaces. Therefore, it can be clearly concluded that there are large number of specific cavities for propranolol adsorption on MIP@WP. Additionally, the imprinting factor (α = QMIP/QNIP) and relative selectivity coefficients (β= αpropranolol/ αanalogue) were calculated to further verify the specific

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recognition for MIP@WP towards propranolol and the results are tabulated in Supporting Information Table S2. It can be obviously seen that MIP@WP had higher imprinting factor than atenolol/pindolol and also relative selectivity coefficients were higher than 1 which also approves the fact that MIP@WP showed excellent adsorption capacity and selectivity towards propranolol.

Regeneration ability has paramount importance for any molecularly imprinted materials for

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further applications. The fabricated molecularly imprinted materials can be regenerated after removing of the template molecule from the polymer network with suitable eluent. In this

case, the adsorption-desorption cycle was performed for 20 times to show the regeneration

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ability and stability of the fabricated MIP@WP. As shown in Fig. 9, after 15 adsorptiondesorption cycles, the maximum adsorption capacity of MIP@WP almost unchanged.

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Meanwhile, the maximum adsorption capacity of MIP@WP declined after more than 15

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adsorption-desorption cycles. This may be the result of deformation of the specific cavities with a large number of adsorption-desorption cycles. Considering all these superior features,

determination.

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the MIP-paper surfaces could be used as a selective sorbent for propranolol separation and

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3.6. Analytical performance of the proposed method

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Analytical performance of the proposed method was determined under optimum conditions. Calibration curve was obtained by plotting absorbance versus different concentrations of propranolol. The obtained calibration curve was linear between 1-1000 µg/mL with an equation y = 0.002x + 0.0337 where y is the absorbance at 290 nm and X is concentration of propranolol (µg/mL) and with correlation coefficient, R2=0.997 (Supporting Information Fig. S1). Limit of detection (LOD), which is expressed as 3Sb/m where Sb is the standard deviation

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of the blank and m is the slope of the calibration curve, was estimated to be 0.30 µg/mL and could meet TDM for propranolol (blood level of propranolol is 6-771 µg/mL [76]). The intraday and inter-day precisions of the proposed method for different spiked concentration levels of propranolol were calculated as being in the range of 2.8%-3.9% (Supporting Information Table S3) indicating that the proposed method is suitable for analytical applications. As clearly seen that the proposed method has obvious advantages such as low cost, short analysis time, simplicity and uncomplicated instrumentation. The proposed method also has acceptable

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linear range and detection limit which is comparable with the blood level of propranolol. As a result, the proposed method can be applied to selective separation and detection of

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propranolol in real sample analysis.

3.7. Application of MIP@WP

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The applicability of the MIP@WP surfaces for selective separation and detection of

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propranolol in plasma samples was tested. For this purpose, plasma samples were spiked with different concentrations of propranolol and adsorption experiments were carried out under optimized conditions. Additionally, the developed method was compared with the traditional

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ELISA method and the obtained results are listed in Table 2. The recoveries of propranolol from the spiked plasma samples varied between 97.0%-99.7% with the relative standard

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deviation below 4.77%, implying that the MIP@WP had high selectivity, accuracy and also

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repeatability when testing plasma samples. Meanwhile, the MIP-@WP exhibited similar sensitivity compared to the traditional ELISA method which also approves that MIP@WP could be applied as a potential alternative, sensitive and selective sorbent for separation and detection of propranolol in plasma samples.

4. Conclusion

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In summary, an analytical method based on MIP@WP and spectrophotometric detection was developed for selective separation and detection of propranolol in plasma samples. The MIP@WP was synthesized via SI-RAFT polymerization in the presence of NAPAL, propranolol, MBAAm and AIBN. The modified paper surfaces were characterized by a combination of several surface analysis techniques such as ATR-FTIR, XPS, SEM and water contact angle measurements. The binding properties were investigated in detail and MIP@WP reached its maximum adsorption capacity (64.3 mg/g) and high imprinting factor (4.20) when

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the initial concentration and adsorption time were kept at 1.0 mg/mL and 60 min at room temperature, respectively. The proposed method was shown to be a sensitive and selective sorbent for separation and detection of propranolol from plasma samples. The major

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advantages of the presented method are easy preparation, low cost, simplicity, rapidity, ease of target separation, high binding capacity and imprinting factor, short extraction time, high

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removal efficiency, high selectivity and low detection limit in terms of TDM for propranolol.

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Moreover, the proposed method not only has common features with the ELISA method, such as sensitivity and selectivity, but also overcomes some limitations of the ELISA method such as excessive washing processes, single use, special equipment and most importantly the

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requirements of antibody-enzyme couple. It is believed that the MIP@WP will enhance the

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applicability of molecular imprinting technology in clinical studies.

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Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this article

Acknowledgements

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This work was financially supported by the Scientific and Technical Council of Turkey

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(TUBITAK), MAG-315M277. AZ also thanks Prof. Dr. Z. Suludere for SEM analysis.

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Figure Captions

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Scheme 1. Representative illustration of the preparation of MIP@WP.

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Fig. 1. ATR-FTIR spectra of (a) pristine WP, (b) BCPA@WP and (c) MIP@WP.

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Fig. 2. XPS wide scan spectra of (a) Pristine WP, (b) BCPA@WP, (c) MIP@WP, and narrow scan spectra of (d) C 1s and (e) N 1s for MIP@WP.

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Figure 3. SEM photomicrographs with low (x100) and high (x5000) magnification, (a) pristine WP, (b) BCPA@WP, (c) MIP@WP, (d) NIP@WP.

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Fig. 4. Water contact angle images of WPs.

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Fig. 5. Effects of (a) amount of propranolol, (b) SI-RAFT polymerization time, (c) type of extraction solvents, (d) washing time.

Fig. 6. (a) Binding isotherms and (b) Scatchard plots for MIP/NIP@WP.

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Fig. 7. (a) Binding kinetics of MIP/NIP@WP, (b) pseudo-first-order and (c) second-orderkinetic models for MIP@WP.

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Fig. 8. (a) Molecular structures of propranolol, atenolol and pindolol, (b) Specific adsorption of propranolol and its analogues on MIP/NIP@WP.

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Fig. 9. Adsorption capacity of MIP@WP after adsorption-desorption cycles.

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Table 1. Effect of monomer type on adsorption capacity and imprinting factor for MIP@WPa. Monomer MAA AAm 4-VP NAPAL

QMIP 23.9 24.6 38.9 64.3

QNIP 9.98 10.2 11.9 14.2

αb 2.39 2.41 3.27 4.20

a

Polymerization was carried under same conditions for each monomer as indicated in Section 2.4

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Imprinting factor, α = QMIP/QNIP

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Table 2. Recovery of propranolol in spiked plasma samples after being separated from MIP@WP (n= 3) Proposed Method ELISA Method Added propranolol (µg/mL) 0

Found (µg/mL)

Recovery (%)

Found (µg/mL)

Recovery (%)

RSD%

-

1.5

1.48 ± 0.06

98.6

4.05

1.49 ± 0.036

99.3

2.42

3.0

2.91 ± 0.11

97.0

3.78

2.99 ± 0.09

99.7

3.01

4.5

4.47 ± 0.22

99.3

4.92

4.51 ±0.15

100.2

3.33

9.0

8.98 ± 0.38

99.7

4.23

8.99 ± 0.25

99.9

2.78

12.0

11.94 ± 0.57

99.5

4.77

11.99 ± 0.31

99.9

2.58

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-

RSD%

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