Synthesis and characterization of a new magnetic restricted access molecularly imprinted polymer for biological sample preparation

Journal Pre-proof Synthesis and characterization of a new magnetic restricted access molecularly imprinted polymer for biological sample preparation ´ Tassia Venga Mendes (Conceptualization) (Methodology) (Investigation) (Writing - review and editing), Lidiane Silva Franqui (Methodology) (Investigation), Mariane Gonc¸alves Santos ´ Wisniewski (Methodology) (Methodology) (Investigation), Celio (Investigation), Eduardo Costa Figueiredo (Supervision) (Investigation) (Writing - review and editing) (Funding acquisition)

PII:

S2352-4928(19)30566-5

DOI:

https://doi.org/10.1016/j.mtcomm.2020.101002

Reference:

MTCOMM 101002

To appear in:

Materials Today Communications

Received Date:

10 May 2019

Revised Date:

7 January 2020

Accepted Date:

12 February 2020

Please cite this article as: Venga Mendes T, Silva Franqui L, Gonc¸alves Santos M, Wisniewski C, Costa Figueiredo E, Synthesis and characterization of a new magnetic restricted access molecularly imprinted polymer for biological sample preparation, Materials Today Communications (2020), doi: https://doi.org/10.1016/j.mtcomm.2020.101002

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Synthesis and characterization of a new magnetic restricted access molecularly imprinted polymer for biological sample preparation Tássia Venga Mendesa, Lidiane Silva Franquib, Mariane Gonçalves Santosa, Célio Wisniewskic Eduardo Costa Figueiredo a* a

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Laboratory of Toxicant and Drug Analyses, Faculty of Pharmaceutical Sciences, Federal University of Alfenas, 37130-000, Alfenas, MG, Brazil b Brazilian Nanotechnology National Laboratory, Brazilian Center for Research in Energy and Materials, 13083-970 Campinas, SP, Brazil c Institute of Exact Sciences, Federal University of Alfenas, 37130-000, Alfenas, MG, Brazil *Corresponding author: Prof. Dr. Eduardo Costa Figueiredo Address: Laboratory of Toxicant and Drug Analyses, Faculty of Pharmaceutical Sciences, Federal University of Alfenas, 37130-000, Alfenas, MG, Brazil Phone number: +55 35 3701 9508 E-mail: [email protected] (E. C. Figueiredo)

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Graphical Abstract

ABSTRACT

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In this paper, a magnetic restricted access molecularly imprinted polymer (M-RAMIP) was proposed as a new sorbent for magnetic dispersive solid phase extraction of small molecules directly from untreated biological fluids. Fe3O4 nanoparticles were synthesized and functionalized with tetraethylorthosilicate and 3- (trimethoxysilyl) propyl methacrylate, resulting in Fe3O4@SiO2-MPS particles. A molecularly imprinted polymer selective to nicotine was synthesized on the Fe3O4@SiO2-MPS surface, resulting in a magnetic molecularly imprinted polymer (M-MIP). Finally, M-MIP particles were encapsulated with a bovine serum albumin (BSA) layer, resulting in the M-RAMIPs. A magnetic restricted access non-imprinted polymer (M-RANIP) was synthesized

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by the same procedure but in absence of the template molecule (nicotine). Adsorption kinetic and isotherm studies were best fitted to the fractional order and Sips models, respectively for kinetic and

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isotherm studies, attesting that the interactions occur by different mechanisms. The M-RAMIP presented higher adsorption capacities in comparison with M-RANIP. This is probably due to the

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presence of selective binding sites in the M-RAMIP. The selectivity test confirmed that the MRAMIP was able to capture more nicotine than cotinine, caffeine, lidocaine, and cocaine in

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comparison with the M-RANIP, however, the covering with BSA reduced the selectivity in comparison with other MIPs from the literature. Protein exclusion capacities of about 79% and 99%

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were observed for M-MIP and M-RAMIP, respectively. M-RAMIP was used to extract nicotine from a human plasma sample, with precision and extraction recovery of about 16 and 27%, respectively. Additionally, the same material was reusable in at least 50 extraction cycles with the

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same performance.

Keywords: restricted access molecularly imprinted polymer, molecularly imprinted polymer, restricted access material, protein exclusion, molecular recognition, nicotine. 1. Introduction

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Molecularly imprinted polymers (MIPs) are selective materials that are able to recognize a

molecule or its analogues [1,2]. The selective binding sites are obtained by fixation of functional monomers in specific positions around a template molecule by using a cross-linker reagent. After the synthesis, the template is removed and the binding sites are exposed. Non-covalent interactions between template molecules and monomers have been the most frequent in MIP synthesis, mainly because they are easily breakable after polymerization. However, the selectivity is not so high compared to the covalent interaction-based MIPs [2]. Temperature can also be a critical parameter 2

for the MIP selectivity, given that a lot of synthesis protocols are carried out at 60oC or higher, whereas the MIP is used in extractions at room conditions. Innovative low temperature synthesis protocols can be highlighted, as for example the molecularly imprinted covalent organic frameworks [3]. MIPs have been often used as sorbents in different sample preparation techniques like conventional solid-phase extraction (SPE) [4,5], solid-phase microextraction [6,7], stir-bar sorptive extraction [8,9], microextraction by packed sorbent [10,11], magnetic dispersive SPE (d-SPE) [12,13], among others. Despite the high selectivity of MIPs, their performance as a sorbent in SPE of untreated

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biological samples have not been very efficient. Proteins from biological fluids can be retained on the MIP surface, causing obstruction of selective binding sites and decreasing the adsorption

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capacity and selectivity [14]. The main approach to solve this problem has been the addition of protective coatings on the MIP surface to avoid protein sorption [15], resulting in restricted access

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molecularly imprinted polymers (RAMIPs). The first strategy to obtain RAMIPs was the fixation of hydrophilic monomers on the external surface of a conventional MIP, as for example glycerol

and

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monomethacylate in association with glycerol dimethacrylate [16,17], glycidyl methacrylate [18,19] 2-O-meth-acryloylozyethoxyl-(2,3,4,6-tetra-O-acettyl-β-D-galactopyranosyl)-(1-4)-2,2,6-tri-

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O-acetyl-β-D-glucopyranoside [20]. The hydrophilic surface acts as a chemical barrier that makes the interaction of RAMIPs with protein from the sample difficult. At the same time, the template molecule can migrate through this hydrophilic layer and access the selective binding sites. Along

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the same line, particles of MIP with surface-grafted hydrophilic polymer brushes prepared by controlled radical precipitation polymerization techniques can be pointed out. These polymers are able to recognize small organic analytes in real, undiluted, biological samples (e.g., milks and serums), without the influence of the macromolecules [21–23]. Another strategy is the use of amphiphilic molecularly imprinted polymers, in which the hydrophobic and hydrophilic groups of

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the monomers are responsible for analyte capture and protein exclusion, respectively [24]. In 2013, Moraes at al.[25] proposed a new biocompatible RAMIP covered with bovine serum

albumin (BSA). The authors observed that the presence of only hydrophilic monomers on the MIP surface was not sufficient to exclude all of the proteins from the samples. Thus, a MIP covered with these hydrophilic monomers was also involved with a BSA capsule chemically crosslinked with glutaraldehyde. The exclusion mechanism occurs when the pH of the medium is higher than the isoelectric point of both proteins from the sample and from the BSA layer. In this case, the proteins 3

will acquire negative charges and be electrostatically repulsed. Exclusion capacities of about 100% have been obtained with RAMIPs covered with the BSA layer, while maintaining their selectivity to the template and analogues [14,26–28]. Other modifications have also been carried out in conventional MIPs to simplify their use in sample preparation. The incorporation of magnetic nanoparticles in the polymer structure is a good example in which the material acquires magnetic susceptibility and it is used in d-SPE, wherein a magnet is employed to remove the particles from samples [29]. An important advantage for using magnetic molecularly imprinted polymers (M-MIPs) in d-SPE is the good interaction between the

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M-MIP and the samples. This gives higher extraction recoveries and good selectivity [30]. Besides, the use of a magnet to remove the sorbent from the sample is a very simple and efficient strategy. It

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avoids problems like the blockage of cartridges, columns, and tubes, which are often faced in conventional SPE. Several applications of M-MIPs for different analytes can be found in the

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literature [12,30–32].

Based on the relevant advantages of the RAMIPs and M-MIPs in terms of the capacity to

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exclude macromolecules, as well as the magnetic susceptibility, respectively, we believe that a new material with both characteristics can be very useful in magnetic d-SPE. In this way, this paper

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reports the development and characterization of a magnetic-restricted access molecularly imprinted polymer (M-RAMIP) selective to nicotine and the investigation of its performance in d-SPE of nicotine and its analogues from human plasma samples. Nicotine was chosen as a proof-of-principle

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drug to appraise the performance of the M-RAMIP. 2. Materials and methods

2.1. Reagents and solutions

Nicotine, cotinine, caffeine, lidocaine and cocaine standards, iron (II) chloride tetrahydrate

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(FeCl2.4H2O), 3- (trimethoxysilyl) propyl methacrylate (MPS), tetraethylorthosilicate (TEOS), methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), 4'-azobis (4-cyanovaleric acid) (ABCVA), methanol, acetonitrile, and BSA were purchased from Sigma Aldrich® (Steinheim, Germany). Iron (III) chloride hexahydrate (FeCl3.6H2O) was obtained from Vetec® (Rio de Janeiro, Brazil). Ammonium hydroxide and isopropyl alcohol were obtained from Isofar ® (Rio de Janeiro, Brazil). Acetic and hydrochloric acids were obtained from Furlab® (São Paulo, Brazil) and Exodus Scientifica (São Paulo, Brazil), respectively.

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2.2. Instrumentation An ultrasonic bath model USC2800A (Unique, São Paulo, Brazil), an mechanical agitator model TE-099 Potter Unit (Tecnal, São Paulo, Brazil), a water bath (Frigomix B) coupled with a Thermomix BM thermostat (B. Braun Biotech International, Melsungen, Germany), a horizontal agitator (Glas-Col, Washington, USA), a vacuum greenhouse (Novatécnica, São Paulo, Brazil), and a heating plate, model NT103 (Novatécnica, São Paulo, Brazil) were used in the synthesis of the polymers. A Vibrax VXR basic tube agitator (IKA®, São Paulo, Brazil), a vortex agitator Lab

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Dancer S25 (IKA®, São Paulo, Brazil), and a neodymium magnet were used in the extraction procedure. An analytical balance (Shimadzu®, Kyoto, Japan) was used for weighing. Ultra-high

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purity water (18.2 MΩ.m) from a Milli-Q system (Millipore®, Bedford, MA, USA) was used in the preparation of solutions. The protein exclusion test was carried out using a high performance liquid

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chromatography system (HPLC) equipped with two Shimadzu LC-20AD pumps (Shimadzu ®, Kyoto, Japan), a manual injector type 7725i (Rheodyne®, Waltham, USA), an electronic six-port

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switching valve model FCV-12AH (Shimadzu®, Tokyo, Japan), and an UV detector model SPD10AVP (Shimadzu®, Tokyo, Japan). An empty cartridge of a guard column (4 mm i.d. x 10 mm)

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was filled with each polymer and used in the protein exclusion test. For the selectivity test, nicotine, cotinine, caffeine, lidocaine and cocaine were analysed using a gas chromatograph mass spectrometer, GCMS QP-2010 from the Shimadzu® (Kyoto, Japan), equipped with a Restek®

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RTX5-MS (30.0 m x 0.25 mm x 0.25 μm) and using helium as drag gas. A transmission electron microscope, JEM-1400 plus (JEOL, USA), a scanning electron microscope, SEM-FEG FEI Inspect F50 (Japan), an X-Ray K-Alpha spectrometer (Thermo Fisher Scientific, USA), a porosimeter Gemini VII version 3.03, model 2390 t (USA), a Fourier transform infrared

spectrometer

(FT-IR),

model IS50

(Thermo

Scientific,

Waltham,

USA),

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thermogravimetric analyser TG-DTA-SDT, Q600 model (TA Instruments, Castle, USA), and an instrument for dynamic light scattering, Zetasizer Nano ZS equipped with MPT-2 Titrator (Malvern, Worcestershire, UK) were used for the characterization studies.

2.3. Preparation of the polymers Fe3O4 magnetic nanoparticles were synthesized by co-precipitation according to Chen et al. [33]. 15 mmol of FeCl3.6H2O and 10 mmol of FeCl2.4H2O were dissolved in 80 mL of deionized 5

water. Afterwards, 50 mL of 28% (v/v) ammonium hydroxide aqueous solution was added into the flask drop by drop, and the reaction was maintained at 80 oC, under agitation and nitrogen atmosphere for 30 min. The obtained black precipitate was washed with deionized water until neutral pH and dried at 60 oC for 24 h. Fe3O4 magnetic nanoparticles modified with TEOS (Fe3O4@SiO2) were obtained according to Zeng et al. [34]. A mass of 600 mg of Fe3O4 magnetic nanoparticles were added in 60 mL of 1:5 (v:v) water:isopropanol solution, and the flask was sonicated during 20 min to promote the nanoparticle dispersion. Then, 10 mL of 28% ammonium hydroxide aqueous solution and 4 mL of

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TEOS were added into the flask with constant agitation for 12 h at 25 oC. Fe3O4@SiO2 nanoparticles were washed with deionized water until neutral pH and dried at 60 oC for 24 h.

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Fe3O4@SiO2 particles modified with MPS (Fe3O4@SiO2-MPS) were obtained according to Kong et al. [35]. A mass of 200 mg of Fe3O4 @SiO2 was dispersed in 50 mL of methanol and

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sonicated for 30 min. Afterwards, 3 mL of MPS was added into the flask, drop by drop, under agitation. The reaction was maintained at 25 oC for 48 h. Fe3O4@SiO2-MPS nanoparticles were

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washed with methanol until the supernatant was completely clear and then dried at 60 oC for 24 h. M-MIPs were synthesized by the precipitation polymerization method, according to Franqui

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et al. [30]. 0.4 mmol of nicotine (template) and 2.0 mmol of MAA (functional monomer) were added into a flask containing 20 mL of acetonitrile; whereas, 489 mg of Fe3O4@SiO2-MPS nanoparticles were added into other flask containing 20 mL of acetonitrile. Both flasks were sonicated for 1 h, and

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the solution of the template and functional monomer was transferred to the flask containing the Fe3O4@SiO2-MPS nanoparticles. 12 mmol of EGDMA (cross linker) and 80 mg of ABCVA (initiator) were also added. The flask was sonicated for 30 min, purged with nitrogen for 15 min, sealed and mechanically stirred for 24 h at 75 oC. The obtained M-MIP was washed several times with 1:9 (v:v) acetic acid:methanol solution followed by washing with pure methanol in order to

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remove the template and other reagents that remained from the synthesis. The M-MIP particles were dried at 60 oC under vacuum for 24 h. The procedure to obtain M-RAMIPs was adapted from Moraes et al. [25]. A volume of 20

mL of 1% (m:v) BSA aqueous solution (prepared in phosphate buffer, 50 mmol L-1, pH 6.0) was added into a tube containing 500 mg of M-MIP. The tube was sonicated for 10 min and maintained in standby for 10 min. Then, the magnetic particles were attracted to the wall of the tube (using a magnet), and the supernatant was discarded. The magnetic particles were then dispersed in 5 mL of 6

25% (m/v) glutaraldehyde aqueous solution and agitation was continued for 5 h. The supernatant was discarded, and the obtained particles were dispersed in 10 mL of 1% (m/v) borohydride aqueous solution under agitation and during 15 min. The obtained M-RAMIPs were separated with a magnet and washed several times with water. A magnetic restricted access non-imprinted polymer (MRANIP) was synthesized by the same procedure used to obtain the M-RAMIP but in the absence of the nicotine (template).

2.4. Characterization of the materials

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Sample dispersions of Fe3O4, Fe3O4@SiO2, M-MIP, and M-RAMIP were prepared in ultrapure water at 100 µg mL-1 and analysed by transmission electron microscopy (TEM). A volume

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of 3.0 µL of each dispersion was applied to an ultrathin lacey Carbon 400 mesh grid; the excess of solution was removed by blotting with a filter paper, and the sample was dried for 30 min. The

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images were recorded at low-dose conditions in order to avoid radiation damage to the samples. The same dispersions (3.0 µL) were also applied to a silicon substrate and analyzed by scanning electron

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microscopy (SEM) (microscope operating at 15 kV). In this case, only the sample containing protein (i.e. M-RAMIP) was previously coated with a thin carbon layer.

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The elemental composition of the sample surface was obtained by X-ray excited photoelectron spectroscopy in survey mode. Data were treated using Thermo Avantage software version 5.957. The obtained results represent the mean of the spectra collected at three different

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points in each sample.

Surface areas of the materials were obtained by using a porosimeter operating with nitrogen gas as adsorbent, an equilibrium time of 10 s, and relative pressure of 0.01 to 0.3. Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-MPS, M-MIP, and M-RAMIP particles were characterized by Fourier transform infrared spectroscopy with horizontal attenuated total reflectance

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(ATR) and employing a zinc selenide crystal. The spectra were obtained in duplicate from 4000 to 500 cm-1 and with a resolution of 4 cm-1 (64 scans). Thermogravimetric analyses were carried out using a thermogravimetric analyser operating

from 25 to 800 °C at a heating rate of 10°C min-1 and under a nitrogen flow of 100 mL min-1. For the zeta potentials studies, 10 mL of 0.01 mg mL-1 M-MIP or M-RAMIP aqueous suspension was titrated using 0.5 and 0.25 mol L-1 NaOH aqueous solutions, as well as 0.25 mol L1

HCl aqueous solution. The study was conducted in the pH range from 3.0 to 10.0. The particle 7

sizes were determined by dynamic light scattering (DLS). For DLS measurements, the attenuation value was 8, and the conductivity of samples ranged from 0.005 to 0.015 mS/cm.

2.5. Adsorption Kinects and isotherm studies The influence of the pH in the adsorption of nicotine on the M-RAMIPs was initially appraised from 4.0 to 10.0. Fractions of 1 mL of 10 mg L-1 nicotine aqueous solution (prepared in phosphate buffers, 0.01 mol L-1, pH 4.0 to 10.0) were placed into 7 different test tubes containing 10 mg of M-RAMIPs. The tubes were agitated using an orbital shaker at 25 ºC during 15 min. A

was

separated.

The

nicotine

concentration

(Ce:

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magnet was put in the wall of each tube in order to attract the magnetic particles, and the supernatant equilibrium

concentration)

was

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spectrophotometrically determined in each supernatant at 260 nm. The equilibrium adsorption capacity – qe (mg g-1) – was obtained according to Equation 1 [36], where Co and Ce (both in mg L) are the initial and equilibrium concentrations, respectively, V (L) is the volume of the solution,

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procedure was applied for M-RANIP. Equation 1: 𝑞𝑒 =

(𝐶𝑜 − 𝐶𝑒 ) ∙𝑉 𝑚

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and m (g) is the mass of the sorbent [36]. The experiment was carried out in triplicate, and the same

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For the kinetic test, 1 mL of 50 mg L-1 nicotine aqueous solution (prepared in phosphate buffer, 0.01 mol L-1, pH 7.0) was placed into 9 different test tubes containing 10 mg of M-RAMIPs. The tubes were agitated using an orbital shaker at 25 ºC during 1, 5, 10, 15, 20, 30, 45, 60 and 105

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min, respectively for each tube. After each time, a magnet was put in the wall of each tube in order to attract the magnetic particles, and the supernatant was separated. The nicotine concentration (Ce) was spectrophotometrically determined in each supernatant at 260 nm using a phosphate buffer solution (0.01 mol L-1, pH 7.0) as a blank. The qe values were determined according to Equation 1. The experiment was carried out in triplicate, and the same procedure was applied for M-RANIP.

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The data were treated according to pseudo-first order, pseudo-second order, fractionary

order, and chemisorption (Elovich) kinetic models [36], taking into consideration the value of the linear correlation coefficient (R2) and the error function (Ferror) (Equation 2) [36] that correlate the amount of analyte adsorbed by the material with that measured experimentally [37]. The number of experiments and parameters of the fitted model are represented by n and p, respectively. 𝑞𝑖,𝑒𝑥𝑝 is each value of 𝑞𝑒 measured experimentally and 𝑞𝑖,𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 is each value of 𝑞𝑒 obtained by the fitted model [36,37]. 8

2

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Equation 2: 𝐹𝑒𝑟𝑟𝑜𝑟 = √(𝑛−𝑝) ∑𝑛𝑖(𝑞𝑖,𝑒𝑥𝑝 − 𝑞𝑖,𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 )

For the absorption isotherm studies, a mass of 10 mg of M-RAMIPs was placed into 10 test tubes containing 1 mL of nicotine aqueous solution (prepared in phosphate buffer, 0.01 mol L-1, pH 7.0) at the concentrations of 50, 100, 200, 350, 500, 800, 1200, 1600, 2000 e 2500 mg L-1. The tubes were agitated using an orbital shaker during 20 min at 25 ºC. A magnet was put in the wall of each tube in order to attract the magnetic particles, and the supernatant was separated. The nicotine concentrations were spectrophotometrically determined in each supernatant at 260 nm, and a

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phosphate buffer solution (0.01 mol L-1, pH 7.0) was used as a blank. The experiment was carried out in triplicate, and the same procedure was applied for M-RANIP. The 𝑞𝑒 values were obtained

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according to Equation 1 and the data were fitted to Langmuir, Freundlich, Sips, Toth and RedlichPeterson models [38]. The best adjustment was obtained using the higher and lower values of R 2

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and Ferror (Equation 2), respectively.

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2.6. Protein exclusion tests

A liquid chromatographic system (HPLC) was used in the protein exclusion test with a

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phosphate buffer solution (0.01 mol L-1, pH 7.0) as the mobile phase (at 1 mL min-1 flow rate). A mini-column (4 mm i.d. x 10 mm) filled with 70 mg of M-RAMIPs was used in the system, replacing the analytical column. A volume of 10 μL of a 44 g L-1 BSA solution (prepared in phosphate buffer,

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0.01 mol L-1, pH 7.0) was injected in the system with the mini-column. The same solution was also injected in the system without any column, and the obtained peak corresponded to 100% of the BSA (all of the injected BSA arrived to the detector). The peak area obtained in the system with the column divided by the 100% BSA peak area corresponds to the tax of BSA excluded by the M-

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RAMIPs [14]. The same procedure was applied for M-MIP.

2.7. Selectivity test

1 mL of a blank plasma sample (from a non-smoker and non-coffee drinker) spiked with 1

mg L-1 of nicotine and one of its analogues (cotinine, caffeine, lidocaine or cocaine) and diluted at a 1:1 (v:v) ratio with ultrapure water was added into a test tube containing 10 mg of M-RAMIPs. The tube was agitated for 40 min at 25 °C. A magnet was used to attract the M-RAMIP particles to

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the wall of the tube. The supernatant was discarded, and the particles were washed twice with 1 mL of water for 10 s. Afterwards, M-RAMIP particles were dispersed in 1 mL of a mixture of methanol and 0.1 mol L-1 hydrochloric acid aqueous solution (ratio 1:0.05, v:v) to elute the molecules. The eluate was separated using a magnet, transferred to another tube (900 µL), and evaporated to dryness under vacuum at 60 °C. Finally, the residue was dissolved in 200µL of methanol and filtered with a 0.22 µm membrane. The experiment was carried out in triplicate and the same procedure was carried out replacing M-RAMIP with M-RANIP. Nicotine, cotinine, caffeine, lidocaine and cocaine were determined in the eluates by gas

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chromatograph mass spectrometry using a Restek® RTX5-MS (30.0 m x 0.25 mm x 0.25 μm) and helium as drag gas at a 1.6 mL min-1 flow rate and with a linear velocity of 47.4 cm s-1. The injector

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was operated at 250 °C in the splitless mode. The following oven programming was used: the initial temperature was 120 °C, followed by increasing to 280 °C at 40 °C min-1, and keeping it at 280 °C

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for 7 min. The total run time was 11 min. The mass spectrometer was operated in the SIM (selective ion monitoring) mode with electron impact ionization (70 eV). The interface and ion source

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temperatures were 280 and 250 oC, respectively. The monitored m/z ratios were 84 and 133 for nicotine, 98 and 176 for cotinine, 109 and 194 for caffeine, 86 and 58 for lidocaine, and 82 and 182

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for cocaine. The quantification was based on the integration of the peak areas of m/z 84, 98, 109, 86 and 182 for nicotine, cotinine, caffeine lidocaine and cocaine, respectively. The adsorptive capacities (𝑞, in µg g-1) of M-RAMIP and M-RANIP for each compound

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were obtained according to equation 1. The binding constant (𝑘) for each material was determined according to Equation 3, where 𝑞𝑡 and 𝑞𝑎 are the adsorptive capacities for the template and the analogue (cotinine, caffeine, lidocaine or cocaine), respectively. Equation 3: 𝑘 = 𝑞𝑡 𝑥 𝑞𝑎−1

The selectivity constants (𝛼) were determined according to Equation 4, where 𝑘𝑀−𝑅𝐴𝑀𝐼𝑃 is

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the binding constant of the M-RAMIP and 𝑘𝑀−𝑅𝐴𝑁𝐼𝑃 is the binding constant of the M-RANIP. −1 Equation 4: 𝛼 = 𝑘𝑀−𝑅𝐴𝑀𝐼𝑃 𝑥 𝑘𝑀−𝑅𝐴𝑁𝐼𝑃

2.8. Precision and recovery 1 mL of a blank plasma sample (from a non-smoker and non-coffee drinker) spiked with 1 mg L-1 of nicotine was diluted at a 1:1 (v:v) ratio with ultrapure water and added into a test tube containing 10 mg of M-RAMIPs. The tube was agitated for 40 min at 25 °C. A magnet was used to 10

separate the M-RAMIP particles, which were washed twice with 1 mL of water for 10 s. Afterwards, M-RAMIP particles were dispersed in 1 mL of a mixture of methanol and 0.1 mol L-1 hydrochloric acid aqueous solution (ratio 1:0.05, v:v) to elute the molecules. The eluate was separated using a magnet, transferred to another tube (900 µL), and evaporated to dryness under vacuum at 60 °C. Finally, the residue was dissolved in 200µL of methanol, and analysed by GC-MS. A 4.5 mg mL-1 nicotine standard prepared in pure acetonitrile was also analysed by GC-MS without any extraction. The experiments were carried out in quadruplicate; the precision of the biological sample analyses was obtained, and the relative standard deviation (RSD) was calculated according to the Equation

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5, were 𝜇 and 𝜎 correspond to the average and standard deviation of the obtained peak areas. 𝜎

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Equation 5: 𝑅𝑆𝐷 = 𝜇 × 100

The extraction recovery was estimated according to Equation 6, were 𝐴1 and 𝐴2 are the peak

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area averages obtained for the nicotine standard solution (prepared in acetonitrile) and plasma sample fortified with nicotine and submitted to the extraction procedure. 𝐴2

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Equation 6: 𝑅 = 𝐴1 × 100

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3. Results and discussions

3.1. Synthesis and characterization of the magnetic restricted access molecularly imprinted polymers

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Fe3O4 magnetic nanoparticles (magnetite) were obtained by the reaction between Fe 2+ and Fe3+ ions in alkaline medium. Afterwards, the treatment of Fe3O4 magnetic nanoparticles with TEOS resulted in Fe3O4 @SiO2, which can be defined as Fe3O4 nanoparticles covered with a silanol polymeric network [39]. The silanol layer is important to decrease the dipolar attraction between the particles, which improves their dispersion capacity. Moreover, this silanol layer is used to anchor

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other chemical groups on the particle surface [40]. In this way, MPS was fixed on the Fe3O4@SiO2 particle surface by the reaction between its silanic groups with the silanols of the particles, resulting in the Fe3O4@SiO2-MPS. On the other hand, the vinyl group of MPS reacted with vinyl groups of methacrylic acid/EGDMA during the polymerization step, promoting the covalent fixation of the MIP on the surface of the Fe3O4@SiO2-MPS particles, resulting in the M-MIP. This material was then encapsulated by a BSA layer through the bonds between the amine groups of the BSA and

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glutaraldehyde which was used as a cross-linker. The obtained imine groups were reduced to amines with NaBH4 in order to improve the chemical and physical stability of the M-RAMIPs [41]. Despite multi-layer grafting (with TEOS, MPS, polymer and BSA) onto Fe3O4 nanoparticles to obtain M-RAMIP particles, the magnetic susceptibility was maintained. As it can be seen in Fig. 1, in about 18 s, most M-RAMIP particles were attracted to the external neodymium magnet

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positioned in the test tube sidewall.

Fig. 1. Use of a neodymium magnet to attract of 100 mg of M-RAMIP particles dispersed in 5 mL

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of water.

Scanning and transmission electron micrographs of the particles are shown in Fig. 2 and Fig.

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3. It was observed that the Fe3O4 particles are in the nanoscale range (<100 nm) (Fig. 2A and Fig. 3A). The increasing in particle size attests to the covering with TEOS (Fig. 2B and Fig. 3B) and with MPS (Fig. 2C). Moreover, a more homogeneous distribution of the magnetic nanoparticles (dark circles) can be observed in the transmission micrograph (Fig. 3B). Clusters of M-MIP microspheres are presented in Fig. 2C and Fig. 3C; these correspond to the MIP synthesis on the

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Fe3O4@SiO2-MPS surface by the precipitation protocol [42]. A more homogeneous external layer can be observed in M-MIP clusters (Fig. 3C) in comparison with Fe3O4@SiO2-MPS (Fig. 3B), probably due to the polymeric network. These clusters were covered with a BSA layer to obtain MRAMIP as represented by Fig. 3D. The porosity of the M-RAMIP can be seen in Fig. 2E and Fig. 2F. Table 1 presents the elemental composition of the particles obtained by X-ray excited photoelectron spectroscopy. The presence of carbon in the Fe3O4 nanoparticles can be explained by

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some possible residual contamination. The presence of silicon in Fe 3O4 @SiO2 and Fe3O4@SiO2MPS attests that the coatings with TEOS and MPS were successful. Additionally, the absence of iron in both materials suggests that the silanol layer is very intense and able to camouflage this metal. The increase in carbon percentages in M-MIP can be explained by the presence of the organic polymeric network. Additionally, the increase of the nitrogen in the M-RAMIP confirms the presence of the BSA layer.

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Table 1: Percentage of iron, oxygen, carbon, silicon, and nitrogen in the elementary surface composition of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-MPS, M-MIP, M-RAMIP Oxygen Silicon Materials Iron (%) Carbon (%) Nitrogen (%) (%) (%) Fe3O4 26.55 45.69 11.24 Fe3O4@SiO2 45.46 10.04 44.50 Fe3O4@SiO2-MPS 44.01 12.63 43.36 M-MIP 26.32 72.49 1.19 M-RAMIP 18.19 67.96 11.92

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Fig. 2. Scanning electron micrographs of the Fe3 O4 (A), Fe3O4@SiO2 (B), Fe3O4@SiO2-MPS (C) M-MIP (D) and M-RAMIP (E and F).

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Fig. 3. Transmission electron micrographs of the Fe3O4 (A), Fe3O4@SiO2 (B), M-MIP (C), and M-

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RAMIP (D).

The superficial areas were estimated by porosity analyses in about 71.93, 12.49, 12.80, 5.28 and 2.86 m2 g-1 for Fe3O4, Fe3O4@SiO2, Fe3O4 @SiO2-MPS, M-MIP and M-RAMIP particles, respectively. A decrease in superficial area was observed when each new layer was inserted on the

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particle surface, attesting to the increase of their dimensions with each covering. M-NIP and MRANIP have presented similar superficial areas in comparison with M-MIP and M-RAMIP, respectively.

Fig. 4 shows the infrared spectra of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-MPS, M-MIP and

M-RAMIP. As it can be seen, the spectrum of Fe 3O4 presents a band at about 550 cm-1, which is characteristic of the stretching vibration of the Fe-O bond (600-550 cm-1) and confirms the existence of iron in the structure of the magnetite [43]. Additionally, the absence of this band (550 cm-1) in

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Fe3O4@SiO2, Fe3O4@SiO2-MPS, M-MIP, and M-RAMIP attests to the Fe3O4 being coated in the subsequent synthesis steps [44]. Bands at 1080, 950, and 800 cm-1 characterize stretching vibrations of the Si-O-Si, Si-O-H and Si-O bonds, and these confirm that the Fe3O4 @SiO2 and Fe3O4@SiO2MPS were coated with TEOS and MPS, respectively [43]. The polymerization reaction on the surface of Fe3O4 @SiO2-MPS particles (forming M-MIP) was confirmed by the bands at 1724 cm-1 as well as those at 1235 to 1147 cm-1 which characterize the vibration of the C=O stretch of esters and the C-O stretching vibration of esters, respectively; all of these were derived from the EGDMA [43]. Bands at 2950 and 1456 cm-1 refer to the stretching of the C-H bond of sp3 carbon as well as

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the asymmetric deformation of this CH3, respectively [43]. No significant differences were observed

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between the M-MIP and M-RAMIP spectra.

Fig. 4. Infrared spectra of Fe3O4 (A), Fe3O4@SiO2 (B), Fe3O4@SiO2-MPS (C), M-MIP (D), and MRAMIP (E).

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Fig. 5 shows the thermogravimetric analyses of the Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-MPS, M-MIP, and M-RAMIP. Weight loss for all the materials at temperatures lower than 200 °C can be attributed to water evaporation. The presence of silanol and vinyl groups in the Fe3O4@SiO2, Fe3O4@SiO2-MPS explain the higher weight losses of both materials (about 11% for both) in comparison with the Fe3O4 (6.10%). M-MIP and M-RAMIP presented similar profiles of weight loss from 400 to 500 oC. This is probably due to the degradation of the organic polymeric network. On the other hand, only M-RAMIP presented weight loss from 200 to 400 oC, which is probably

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due to the degradation of the BSA layer [45].

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Fig. 5. Thermogravimetric analyses of the Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-MPS, M-MIP, and MRAMIP. Fe3O4@SiO2 and Fe3O4@SiO2-MPS profiles are overlapped. The zeta potential curves of M-MIP and M-RAMIP are presented in Fig. 6. The isoelectric points (IP) of M-MIP and M-RAMIP were 4.6 and 4.8, respectively, indicating the presence of the BSA layer on the M-RAMIP. It is important to point out that the isoelectric point of pure BSA ranges

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from 4.7 to 4.9, according to the literature [46]. Additionally, M-MIP and M-RAMIP presented positive or negative charges for pH < IP and pH>IP, respectively. The hydrodynamic diameters obtained by DLS were 641.6, 530.3, 672.6 and 561.72 nm, for M-MIP, M-NIP, M-RAMIP and MRANIP, respectively. As it can be seen, the imprinted particles were larger than the non-imprinted particles, probably due to the formation of the selective binding sites involving the template molecule. Additionally, larger particles were obtained for the covered materials (M-RAMIP and MRANIP) due to the presence of the BSA external layer.

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Fig. 6. Zeta potential of the M-MIP and M-RAMIP.

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3.2. Kinetic and thermodynamic adsorption studies

The adsorption pH was appraised from 4.0 to 9.0 and the highest adsorption capacity (qe=0.5 mg g-1) was obtained for pH 7.0. At this pH, the methacrylic acid of the polymeric network

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(pka=4.25) and nicotine (pka=8.5) are negatively and positively ionized, respectively, and the interaction between both molecules occurs by electrostatic attraction, justifying the highest

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adsorption capacity. Extraction efficiencies of about 0.3 and 0.4 mg g-1 were obtained at pH 4.0 and 9.0, respectively, attesting that weaker bonds prevail in both cases. The same behaviour can be observed for M-RANIP due to its similar chemical composition in comparison with the M-RAMIP.

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In this way, pH 7.0 was adopted as the working condition for the next experiments. As it can be seen in Fig. 7A, the adsorption equilibrium was reached in about 20 min for both materials in the adsorption kinetic studies. Additionally, the adsorptive capacities of the MRAMIP was highest in comparison with the M-RANIP. This probably indicates the presence of

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selective binding sites in the M-RAMIP. The kinetic data were adjusted to the pseudo-first order, pseudo-second order, fractional order, and chemisorption (Elovich) kinetics models, and the best fit was obtained for the fractional order model, according to the highest values of R2 and low values of Ferror, respectively; this can be seen in Table 2. These results indicate that instead of the adsorption mechanism following only a simple kinetic order, the adsorption process followed kinetics of multiple orders that are altered during the contact between the adsorbate and adsorbent [47,48].

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Table 2: Kinect parameters for the nicotine adsorption on M-MIP, M-NIP, M-RAMIP, and M-RANIP fitted to the pseudo-first order, pseudo-second order, chemisorption, and fractionary order models. Value obtained Kinetic model Equation Parameter M-RAMIP M-RANIP qe (mg g-1) 3.16286 2.73982 -1 k1 (min ) 0.86963 0.24161 Pseudo-first order 𝑞𝑡 = 𝑞𝑒 [1 − 𝑒𝑥𝑝(−𝑘1 𝑡)] 2 R 0.97272 0.9479 Ferror 0.00014624 0.049575308 -1 qe (mg g ) 3.31428 2.91407 -1 -1 2 𝑘2 𝑞𝑒 𝑡 k2 (g mg min ) 0.36988 0.15517 Pseudo-second 𝑞𝑡 = 2 order R 0.99624 0.98298 1 + 𝑘2 𝑞𝑒 𝑡 Ferror 5.03427E-05 0.002694133 -1 -1 α (mg g min ) 334.78382 10.11368 -1 1 1 β (g mg ) 3.2696 2.51881 Chemisorption 𝑞𝑡 = 𝐿𝑛 (𝛼𝛽) + 𝐿𝑛 (𝑡) 2 (Elovich) R 0.98027 0.96915 𝛽 𝛽 Ferror 4.10826E-10 1.8304E-12 qe (mg g-1) 3.33596 2.89298 -1 kAV (min ) 0.67648 0.2375 Fractionary order 𝑞𝑡 nAV 0.43376 0.54539 (Avrami) = 𝑞𝑒 {1 − 𝑒𝑥𝑝[−(𝑘𝐴𝑉 𝑡)]𝑛𝐴𝑉 } R2 0.9992 0.99412 Ferror 0.127354108 0.573845471 qt: amount of analyte adsorbed at time t; qe: amount of analyte adsorbed at equilibrium per gram material; t: time of contact; k1: pseudo-first order rate constant; k2: pseudo-second order rate constant; α: initial adsorption rate of Elovich equation; β: Elovich constant related to the extent of surface coverage and also to the activation energy involved in chemisorption; kAV: Avrami kinetic constant; nAV: fractionary reaction order (Avrami) related to adsorption mechanism.

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Fig. 7. Nicotine adsorption kinetics (A) and isotherms (B) in the M-RAMIP and M-RANIP.

Fig. 7B shows the results of nicotine thermodynamic adsorption tests in M-RAMIP and M-

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RANIP, and the higher adsorption capacities for M-RAMIP may be due to the presence of selective binding sites. The data were submitted to the Langmuir, Freundlich, Sips, Toth and Redlich-

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Peterson models, and the obtained parameters are shown in Table 3. The best adjustments can be observed for Sips and Toth models in terms of high and low values of R2 and Ferror, but there is an

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advantage for the Toth model, in terms of R2. The Toth model is a modification of the Langmuir equation and describes heterogeneous materials that adsorb molecules in multi-layers. [38]. However, unlike other models, the lowest energy sites are those with the highest adsorption [38].

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An advantage of the Toth model is that it can be applied to systems with wide concentration ranges, as in this paper.

3.3. Protein exclusion test

The abilities of M-MIP and M-RAMIP to exclude macromolecules were investigated and

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the results are presented in Fig. 8. About 21.4 % of the proteins injected through the M-MIP columns were retained. On the other hand, the M-RAMIP column was able to exclude about 100% of the proteins that flowed through it. Thus, the BSA layer of the M-RAMIP acts as an important biocompatible film on the magnetic polymer. The exclusion mechanism is probably based on the electrostatic repulsion between the proteins from the solution and from the BSA layers of the MRAMIP; both of these are negatively charged at pH 7.0 [27].

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Table 3: Isotherm parameters for nicotine adsorption on M-MIP, M-NIP, M-RAMIP, and M-RANIP fitted to the Langmuir, Freundlich, Sips, Redlich-Peterson, and Toth models. Obtained value Isotherm Equation Parameters model M-RAMIP M-RANIP qs (mg g-1) 10.36477 8.70592 𝑞𝑆 𝐾𝐿 𝐶𝑒 KL (mg L-1) 0.08275 0.05421 Langmuir 𝑞𝑒 = 2 R 0.98233 0.94531 1 + 𝑏𝐶𝑒 Ferror 0.000121068 0.09989669 KF (mg g-1) (mg L-1) 1.36579 0.77449 1⁄ n 2.01796 1.7735 F 𝑛 Freundlich 𝑞𝑒 = 𝐾𝐹 𝐶𝑒 𝐹 R2 0.93645 0.89058 Ferror 0.324673928 0.320999798 -1 q S (mg g ) 9.30828 6.5347 1⁄ -1 𝑛𝑆 K (mg L ) 7.056E-02 2.33E-02 S 𝑞𝑆 𝐾𝑆 𝐶𝑒 Sips 𝑞𝑒 = n 1.17445 1.5902 S 1⁄ 𝑛𝑆 2 R 0.98277 0.9577 1 + 𝑎𝑆 𝐶𝑒 Ferror 0.10486859 0.204634832 -1 KR (g L ) 0.85765 0.47191 𝐾𝑅 𝐶𝑒 𝑞𝑒 = aR (mg L-1) 0.08275 0.05421 𝑔 Redlich1 + 𝑎𝑅 𝐶𝑒 g 1.0000 1.0000 Peterson R2 0.98036 0.93923 where 0 ≤ g ≤ 1 Ferror 0.000152112 0.104337628 -1 KT (mg g ) 51.05534 4119.0324 -1 a (L mg ) 81.06317 13598.4402 T 𝐾𝑇 𝐶𝑒 𝑞𝑒 = Toth t 0.70611 0.38276 1 (𝑎 𝑇 + 𝐶𝑒 ) ⁄𝑡 R2 0.98765 0.98072 Ferror 0.148015177 0.014654604 qe: Amount of adsorbed analyte at equilibrium per gram of material; qs: Theoretical saturation capacity; KL: Langmuir affinity constant; Ce: Analyte concentration at equilibrium; KF and nF: Constant and exponent of Freundlich model, respectively; Ks and ns: Constant and exponent of Sips model, respectively; KR and aR: Constant and g is exponent of Redlich-Peterson model, respectively; KT and aT: Constants isotherm of Toth model; t: Exponent of Toth model.

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Fig. 8. Analytical signals obtained by the injection of 10 µL of the 44 mg mL -1 BSA solution

system with the M-MIP or M-RAMIP columns.

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3.4. Selectivity test

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(prepared in phosphate buffer, 0.01 mol L-1, pH 7.0) into the system without a column or into the

The best way to appraise the selectivity of an imprinted material is by investigating its

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adsorption capacity for the template as well as for other molecules. In this case, nicotine (template), cotinine, caffeine, lidocaine, and cocaine were used. Binding (𝑘) and selectivity constants (𝛼) for the nicotine/cotinine, nicotine/caffeine, nicotine/lidocaine and nicotine/cocaine mixtures are

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shown in Table 4. As it can be seen, the M-RAMIP presented the highest adsorption capacity for nicotine in comparison with cotinine, caffeine, lidocaine and cocaine. Thus, the selectivity constants for nicotine/cotinine, nicotine/caffeine, nicotine/lidocaine and nicotine/cocaine were 1.58, 1.40, 4.25, and 1.47, respectively. This result attests to the selectivity of molecularly imprinted material to the template molecule. Lidocaine were less retained probably because is the

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most lipophilic molecule in comparison with cotinine, caffeine, and cocaine. A similar selectivity constant (about 1.66) was obtained by Zhou et al. for the simultaneous interaction of nicotine/cotinine in a conventional MIP selective to nicotine [49]. This fact confirms that, even with the covering of the M-MIP with the BSA layer, the selective interactions prevail. This is probably due to the migration of the molecules through this layer and their interaction with the selective binding sites in the molecularly imprinted core. Other papers have reported higher selectivity constants for conventional MIPs in comparison with the M-RAMIP proposed in this 22

work [50], probably due to the presence of the BSA external layer in M-RAMIP, which can also retain different molecules by unspecific interactions. Despite this limitation, the ability of excluding macromolecules (obtained for MIPs covered with BSA) need to be highlighted as an important advantage in inducing the use of this sorbent in biological sample preparation. The MIP proposed in this paper was not submitted to the selectivity tests because of its low efficiency for excluding protein. These sorbed proteins can be eluted from MIP and inserted into the GC-MS equipment, dandifying it. Finally, for simultaneous chromatographic analysis of different molecules, it is more

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Table 4. Binding constants (𝑘) and selectivity constants (𝛼) for the nicotine/cotinine, nicotine/caffeine, nicotine/lidocaine and nicotine/cocaine mixtures. Binding constanti Selectivity Mixture constantii M-RAMIP M-RANIP Nicotine/cotinine 5.87 3.71 1.58 Nicotine/caffeine 1.73 1.24 1.40 Nicotine/lidocaine 3.91 0.92 4.25 Nicotine/cocaine 1.34 0.92 1.47 i Binding constant obtained by dividing the nicotine adsorption capacity by the cotinine, caffeine, lidocaine or cocaine adsorption capacity; iiselectivity constant obtained by dividing the binding constants for M-RAMIP and M-RANIP.

3.5. Precision, recovery and reusability

The precision (RSD) for the extraction of nicotine from a human plasma sample fortified with nicotine at 1.0 mg L-1 was about 16%. This RSD value is already suitable for bioanalyses

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according to the USA Food and Drug Administration [51]. Additionally, we are sure that the use of a deuterated internal standard may further reduce the RSDs. The obtained extraction recovery was about 27% and this corresponds to the amount of

nicotine that was removed from the plasma sample during an extraction cycle. In fact, conventional MIPs described in the literature have presented extraction recoveries higher than 50% [52].The relatively low extraction recovery of the proposed material can be attributed to the presence of the BSA external layer, which decreases the extraction capacity by obstructing of 23

some binding sites of the imprinted polymer [25]. Despite this limitation, it is important to point out that the material can be used in bioanalytical applications if the biological curve is prepared in a fortified plasma standard submitted to the same extraction procedure as the real samples. Additionally, the same M-RAMIP particles were used in at least 50 sequential offline dSPE with the same performance, attesting to their good lifetime. A regeneration step was not necessary in this case. On the other hand, the use of columns packed with M-RAMIPs in online extractions was not satisfactory. The presence of the BSA layer, results in a gradual compression of the material in the columns interior, being extremely difficult to flow a liquid (water or organic

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BSA [53].

4. Conclusions

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An M-RAMIP was successfully synthesized and characterized, and its selectivity to nicotine was confirmed in comparison with cotinine, caffeine, lidocaine and cocaine. The main

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limitations of M-RAMIPs are: i) selectivity was not as high as in conventional MIPs (uncovered with BSA), probably because different molecules may be non-specifically retained by the BSA

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layer; and ii) the extraction recovery was not as high as in conventional MIPs, probably because the BSA layer obstructed some selective binding sites. Despite these limitations, the presence of the BSA layer was very important to avoid the retention of proteins from the sample on the M-

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RAMIP surface, being possible to extract nicotine from human plasma samples without any previous treatment. M-RAMIP also presented a good interaction with the external magnet; it was quickly attracted to it, being adequate to be used in d-SPE of nicotine directly from untreated biological samples, such as blood, plasma, serum, and milk. The same synthesis protocol can also

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be used to obtain M-RAMIPs selective to other molecules. Author statement

Tássia Venga Mendes: Conceptualization, Methodology, Investigation, Writing- Reviewing and Editing, Lidiane Silva Franqui: Methodology, Investigation Mariane Gonçalves Santos: Methodology, Investigation Célio Wisniewski: Methodology, Investigation Eduardo Costa Figueiredo: Supervision, Investigation, Writing- Reviewing and Editing, Funding acquisition

Conflicts of interest There are no conflicts to declare. 24

Acknowledgements The authors are thankful to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte, Brazil), projects CDS-APQ-00638-17, CDS-PPM-00144-15 and CEX-APQ-01556-13; to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil), project 483371/2012-2; and to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasilia, Brazil) for their financial support.

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Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

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[25]

[26]

[30]

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