Selective Sorption of Uric Acid by Novel Molecularly Imprinted Polymers

Molecular Imprinting Research Article • DOI: 10.2478/molim-2012-0003 • MOLIM • 2013 • 17–26

Selective sorption of uric acid by novel molecularly imprinted polymers

Abstract Anastasia P. Leshchinskaya*, Oleg A. Pisarev, The polymers based on ethylene glycol dimethacrylate (EGDMA) and Irina V. Polyakova, Evgeniy F. Panarin dimethylaminoethyl methacrylate (DMAEMA) and molecularly imprinted Anna R. Groshikova, with uric acid (UA), UA-MIPs, were successfully synthesized. The binding activity of UA-MIPs towards UA was studied in depth using batch methods. Department of Biopolymers, The optimized sorbent UA-MIP-7-16 was synthesized; this is an EGDMA- Institute of Macromolecular Compounds, crosslinked system containing 16 mol% of UA as the template. The Russian Academy of Sciences, character of binding between UA and UA-MIP-7-16 was studied using Saint-Petersburg, 199004, xanthine as a reference substance, since its chemical structure is similar Bolshoi pr. 31, Russia to that of UA. The studies of equilibrium sorption of UA and xanthine from model aqueous solutions by the imprinted sorbent demonstrate the predominance of specific UA sorption. The sorption kinetic data were analyzed using the Boyd model and shell and core. Selectivity of UA- MIP-7-16 was further demonstrated by biochemical analysis of serum containing UA and other components conducted before and after sorption. UA-MIP-7-16 showed high recognition selectivity and affinity towards the template molecule (UA).

Keywords Molecularly imprinted sorbents • Uric acid • Selective sorption • Serum

Received 20 April 2012 © Versita Sp. z o.o. Accepted 10 July 2012

1. Introduction selectivity. Besides, there are other significant requirements, e.g. biocompatibility, capability to biodegrade and hemocompatibility Uric acid (UA) is the primary product of purine degradation in [11,12]. humans and can serve as a marker of uremic toxins. UA level Molecularly imprinted polymers (MIPs) are artificial sorbents in serum is an important index for assessing renal function possessing high selectivity towards the target molecule present and identifying a variety of kidney diseases [1-3]. The UA in multicomponent mixtures. The most common strategy in concentration in serum exceeding 420 µmol/L can provoke MIP preparation consists in using interactions between the hyperuricemia, gout, chronic renal failure, Lesch-Nyan disease target molecule (template, imprint) and some functional groups. and other disorders in human organism. Besides, UA is a factor These interactions lead to the formation of the complexes in the metabolic syndrome [4,5]. Currently, there are two ways between template and functional monomer in solution. During for treating hyperuricemia, chronic renal failure and gout: special polymerization, target templates are mixed with functional diet which is very often ineffective and drug therapy having monomers. After the formation of polymer, template molecules undesirable side effects [6,7]. are extracted from polymer network. As a result, specific Uric acid accumulated in blood can be removed effectively recognition cavities are formed [13,14]. using blood purification techniques in order to attenuate There are two strategies for molecular imprinting; in the first toxicosis symptoms in patients [8-10]. It is an attractive research method, template is bound with functional monomers by non- subject in the areas of chemistry and medicine. The attempts covalent interactions, and in the second one, covalent bonds are being made to create polymeric sorbents possessing high are used [15,16]. Nowadays, polymers imprinted with different selectivity and sorption capacity; these sorbents may be used templates like drugs, herbicides, sugars, nucleotides, amino to remove uremic toxins directly from blood. A lot of sorbents acids and proteins are widely used in analytical science, as for hemoperfusion were used to remove small molecular well as in catalysis and synthesis [17,18]. Moreover, MIPs have weight products of endogenous catabolism such as UA. a considerable potential for application in the areas of clinical However, the studied sorbents have low sorption capacity, analysis, medical diagnostics, environmental monitoring and and it is more important that none of these sorbents possess drug delivery. MIPs are easy to prepare, stable, inexpensive and

* E-mail: [email protected]

17 A.P. Leshchinskaya et al.

capable of molecular recognition [19,20]. We have developed a loading solution into column. The fractions were collected using number of original methods of controlling sorption equilibrium an 1220 fraction collector (ISCO, USA). and selectivity of sorption of biologically active target substances by polymeric sorbents [21-23]. 2.2. Preparation and characterization of UA-MIPs. In this study, our principal objective was to synthesize a The UA-MIPs were synthesized according to the standard number of UA-MIPs. The second goal was to investigate the procedure with a few modifications; the steps of the synthesis capability of UA-MIPs for the selective recognition of UA from are listed below. model solutions. The selective sorption capacity of UA-MIP (a) Preparation of pre-assembly solution. UA was used as a (an effective indicator of selective sorption) was established template molecule and added to the copolymerization mixture, as as the difference between sorption capacities of UA-MIP and well as soluble salts of UA and different organic bases (Table 1). non-imprinted polymers (NIP). The kinetics and dynamics of UA a selective sorption were also investigated. Further, the selective recognition ability of the novel MIPs was evaluated using serum with high concentration of UA. In the preparation of the novel MIPs, the following compounds were used: UA as a target molecule, DMAEMA as a functional monomer and EGDMA as a crosslinker. The corresponding NIPs were synthesized under the same synthesis conditions, but without UA. b

2. Materials and methods

2.1. Materials and instruments Chemically pure uric acid (2,6,8-trioxypurine) and xanthine (2,6-dioxypurine), Vekton, Russia, were used in experiments. The formulas of UA and xanthine are shown in Figure 1. Figure 1. Tautomeric forms: a) uric acid; b) xanthine. Chemically pure DMAEMA and EGDMA (Acros Organics, Belgium, Figure 2) were used in polymerization. a Peritoneal liquid obtained from patients after dialysis was used as the solution which models serum composition most adequately. We have also used serum with high concentration of UA (more than 420 μmol/L), which was obtained from people with chronic renal failure and gout. Single-component water solutions of UA (or xanthine) were used for studying the main sorption parameters. Since UA is low b soluble in water, it was dissolved in solution of Li2CO3 (0.3 g/L). Xanthine was dissolved in the 0.1 N solution of NaOH. The optical density measurements were performed using an SPH-256 spectrophotometer (LOMO, Russia). The sorption dynamics experiments were carried out using laboratory columns of different sizes. An PP-1M pump (LOMO, Russia) was used for Figure 2. Structural formulas: a) DMAEMA; b) EGDMA.

Table 1. Characteristics of UA-MIPs.

ECN+, ECCOO-, Sorbent Salt UA-organic base DMAEM EGDMA mass yield % Кs ρ, g/ cm3 mg-eq/g mg-eq/g

UA-MIP-1-0.08 UA- Li2 CO3 75 25 3.3 1.7 85 1.7 0.68 UA-MIP-2-16 UA-guanidine 20 2 2.8 – 98 1.1 0.49

UA-MIP -3-16 UA-diallylamin 70 30 4.4 – <30 2.5 0.37

UA-MIP-4-16 UA-diallylamin 46 54 2.6 0.4 <30 1.7 0.6

UA-MIP-5-7 UA-ethylenediamine - 100 - - 93 1.1 0.53

UA-MIP-6-16 UA-diethylamine - 100 - - 84 1.05 0.56

UA-MIP-7-16 UA-diethylamine - 100 - - 84 1.2 0.47

18 Selective sorption of uric acid by novel molecularly imprinted polymers

Thus, UA was dissolved in diallylamine, ethylenediamine, 2.3. Batch studies of equilibrium diethylamine and guanidine. The sorption equilibrium parameters were studied statically. The (b) Preparation of pre-polymerization solution. UA salt experiments involving UA sorption were carried out as follows: (4 wt%) was added to 100 mL of glycerin and stirred for 15 min. 10 mL of model solution with the known UA content was added Then, the pre-assembly solution was added to the mixture of into the bottle with 10 mg of swollen sorbent. The solution was EGDMA (20 wt%) and DMAEMA (2 wt%) and stirred for 10 min stirred with the sorbent for 24 h until the equilibrium was reached. to prepare the pre-polymerization solution. UA concentration in the equilibrium single-component aqueous (c) Polymerization. The pre-polymerization solution was solution was calculated from optical density of this solution at poured into a three-necked round-bottomed flask, and 0.08 g of 293 nm with the aid of calibration curve. Similar experiments azobisisobutyronitrile (AIBN) was added. The mixture was stirred in were also carried out during investigation of xanthine sorption

N2 atmosphere, while the temperature was increased up to 70°C. equilibrium. The reaction was carried out at a temperature of 70°C for 2 h. UA concentration in peritoneal liquid was determined using (d) Processing of products. After polymerization, the product the assay kit “Uric acid. Determination of concentration of uric was washed with a mixture of hot water and ethanol (1:1); the acid in serum/plasma by enzymatic colorimetric method without presence of residual low molecular weight compounds was deproteinization” (Vital diagnostics, Russia). Two main reactions detected using UV-spectr (peaks at 210 nm for diethyl amine can describe the concept of this method: and at 293 nm for UA). Template UA molecules were extracted from polymer network with the mixture of 0.1 N HCl solution and uricase URIC ACID + 2Н2О + О2 – → allantoin + СО2 + Н2О2 ethanol (1:1) in the Soxhlet apparatus for 5 h until the template peroxydase 2 Н2О2 + DCPS + ААP – → (dyed complex of quinoneimine) +4Н2О molecule could not be detected by UV spectrophotometer. Then the polymers were again washed with water three times and where DCPS is 3,5-dichloro-2-phenolsulfonate; ААP is dried at 50°C. 4-aminoantipyrine. (e) The corresponding non-imprinted polymers (NIPs) were In the first stage, UA is oxidized by uricase with the formation prepared in a similar way to that described above, but without of Н2О2. Then, Н2О2 is oxidized by chromogens with the formation the addition of UA. NIPs were used as reference samples during of dyed complex. The intensity of IR band at 520 nm is directly characterization. proportional to the concentration of uric acid in peritoneal liquid. Then, sorbents were dried to constant weight. The sorbent The concentration of UA was calculated using the equation: particles with a size of 160-315 µm were obtained by grinding and sifting. (3) С=(Еtt/Еct)×357 μmol/L

The swelling coefficient К( s) was defined as a ratio between volumes of swollen and dry MIPs: where С is the concentration of UA in peritoneal liquid

(μmol/L), Еtt is the IR absorption intensity at 520 nm in test (1) tube, Е is the IR absorption intensity at 520 nm in control tube, Кs = Vs / Vd ct 357 μmol/L is the concentration of UA in control tube. where V is the volume of swollen sorbent (cm3), V is the The sorption capacity was calculated using the equation: s d volume of dry sorbent (cm3).   The packing density of MIPs (ρ, g/cm3) was calculated as: (C C eq ) V (4) Qe   1000 m s , (2) where С, С are the initial and equilibrium concentrations of ρ = ms / Vs eq UA, respectively (mmol·mL-1), V is the volume of solution (mL), 3 where V is the volume of solution (cm ), ms is the mass of ms is the mass of sorbent (mg). sorbent (g). We have also determined the coefficient of distribution of The total exchange capacity (EC, mg-eq/g) of MIPs was UA (or xanthine) between mobile phase and stationary phase of determined for amino- and carboxylic groups. For titration sorbent as: of MIP amino groups, sorbents were previously converted to OH- form. The experiments were carried out as follows: 20 mL (5) Γi = Qe·ρs/ Кs·Ceq of 0.1 N HCl was added into the bottle containing 10 mg of sorbent. The solution was stirred for 7 days. Then, aliquot was where Qe is the sorption capacity of sorbent for UA (mg/g), taken and titrated with 0.1 N NaOH using methyl orange as an is the packing density (g/mL), is the swelling coefficient,C ρs Кs eq - -1 indicator, and ECCOO was calculated. For titration of sorbents, is the equilibrium concentration of UA (mmol·mL ). carboxylic groups of polymer were previously converted to H+ form. Then, 20 mL of 0.1 N NaOH was added into the bottle 2.4. Thermodynamics of sorption containing 10 mg of sorbent. The solution was stirred for 7 days; The free energy of sorption ΔG (J/mol) was calculated as: then, aliquot was taken and titrated with 0.1 N HCl using methyl + orange as an indicator, and ECN of MIP was calculated. ΔG = - RTlnΓ (6)

19 A.P. Leshchinskaya et al.

where R is the universal gas constant (8.31 J·-1·К-1), T is the The average time of sorption was calculated as: temperature (К), lnГ is the logarithm of distribution coefficient. The enthalpy of sorption ΔH (J·mol-1) was calculated as: t =L2(1 + 3ρ + 6 ρ2 + 5 ρ3)/15 D (1 + ρ + ρ 2 ) (13)

ln Г 2  ln Г1 (7) where: is the relative “core radius”, which is calculated by ΔH = - R ρ 1/Т 2 1/Т 1 means of the following formula: where T , T are the temperatures 293 К and 310 К, 1 2 R  L respectively, lnГ and lnГ are the logarithms of distribution   (14) 1 2 R coefficients at 293К and 310 К respectively. The value of Г was calculated by integration of function Thickness of the sorption layer (L) was determined , where is the degree of saturation of sorbent with sorbate. experimentally as the average radius of sorbent particles which Гi =f(γ) γ The degree of saturation (γ) was calculated as a relationship provides the maximum UA sorption capacity. The method of between the sorption capacity at the i-point of the equilibrium grinding with subsequent sieving and suspension deposition isotherm and the maximal possible sorption capacity. yielded the following fractions of sorbent particles: 8 ÷ 24 μm, The entropy ΔЅ (J·mol-1·К -1) was defined as: 25 ÷ 60 μm, 71 ÷ 80 μm, 80 ÷ 100 μm, 100 ÷ 160 μm, 160÷ 315 μm, 315 ÷ 400 μm, 400 ÷ 500 μm, 500 ÷ 800 μm. Then, ΤΔЅ= ΔH - ΔG. (8) UA equilibrium sorption experiments were conducted using each fraction. The average grain radius which provides the maximum 2.5. Sorption kinetics of uric acid UA sorption capacity was taken as a thickness of the sorption The experiments on kinetics of UA sorption were carried out as layer L [24,25]. follows: 50 mL of model solution with the known content of UA was added into the bottle containing 50 mg of swollen sorbent. 2.6. Dynamics of uric acid sorption In the course of experiment, during continuous stirring, samples The frontal dynamic sorption experiments were conducted on (0.2 mL) were taken at defined intervals. Then, the concentration the laboratory columns: 30 mL of the UA model solution with of UA in samples was determined from optical density of the UV the known initial concentration was loaded into the column with absorption band at 293 nm with the use of calibration curve. the studied sorbent. After sorption, the concentration of UA at The kinetics of sorption of UA by UA-MIP-7-16 was the outlet of the column was determined spectrophotometrically investigated using two kinetic models: (a) the Boyd model and by measuring optical density at 293 nm. (b) the “shall and core” model [24]. Elution curves, С = f (V), were obtained, where С is the The Boyd model is commonly used for description of concentration of UA in eluate (µmol/L); V is the elution volume (mL). intraparticle diffusion mechanism of mass transfer. This model assumes homogeneous distribution of sorbate. 3. Results and discussion

2 2 D= 0,087 tgβ R (9) 3.1 Synthesis and design of MIPs. Selection of the best

MIP. where D is the diffusion coefficient, cm2∙s-1; R is the radius The synthesis of new crosslinked polymers based on DMAEMA of the swollen sorbent, μm; β is the slope of linear part of the as a functional monomer and EGDMA as a crosslinker was F = f(t1/2) dependence, where F is the degree of equilibrium studied. The study of the main physico-chemical and sorption reached at the time t. properties of the sorbents showed the influence of the DMAEMA The average time of sorption was calculated as: hydrolysis activity on the formation of polyampholyte matrix of 1 polymers. The composition of each MIP is shown in detail in t   f (t)dF (10) Table 1 . 0 UA-MIP-1-0.08 are polyampholytes; they can absorb UA In the case of intradiffusion kinetics: only from model aqueous solutions (Table 2). These polymers R2 were synthesized with the addition of 0.08 mol% of UA to the t  15D (11) polymerization mixture. According to “shell and core” model, kinetic parameters To optimize the performance of introduced uric acid as of sorption are calculated taking into account sorption of a a template molecule, UA was dissolved in different organic substance in a limited layer of sorbent. bases, which cannot act as monomers in the polymerization. Thus, UA was dissolved in guanidine (16 mol%), and UA- 1 6 Dt F  MIP-2-16 was synthesized. UA-MIP-2-16 is an anionic L  LL   (12) polymer, demonstrates low rate of swelling (К = 1.1), the 3 3   s  RR  synthesis proceeds with a good mass yield (85%), and the where L is the sorption layer, μm; t is the average time of polymer can absorb UA only from model aqueous solutions diffusion. (≈ 70 mmol/g).

20 Selective sorption of uric acid by novel molecularly imprinted polymers

Table 2. Sorption equilibrium of UA by UA-MIPs from aqueous solution and peritoneal liquid.

Equilibrium sorption of UA from water Equilibrium sorption of UA from peritoneal Sorbent solution Qe, mmol/g liquid Qe, mmol/g

1 UA-MIP-1-0.08 100 -

2 UA-MIP-2-16 70 -

3 UA-MIP -3-16 120 -

4 UA-MIP -4-16 100 10

5 UA-MIP -5-7 40 -

6 UA-MIP -6-16 160 40

7 UA-MIP -7-16 180 70 a b

Figure 3. Equilibrium sorption of UA from water solution by UA-MIP-7-16 and NIP with 0.9% NaCl and without NaCl.

Then, UA was dissolved in diallylamine, and UA-MIP-3-16 3.2. Equilibrium sorption of UA from model solutions and UA-MIP-4-16 were synthesized. UA-MIP-3-16 is an anionic To study the UA sorption specificity, we used two sorbents: polymer, it can absorb UA only from the model aqueous UA-MIP-7-16 and its NIP. The equilibrium sorption of UA and solution. UA-MIP-4-16 is a polyampholyte, it can absorb UA its closest structural analogue (xanthine) from model aqueous both from model aqueous solutions and peritoneal liquid (10 solutions and complex peritoneal liquid was studied. mmol/g). The mass yields of these sorbents were low (<30%). First, adsorption experiments using the batch method were Modification of the synthesis conditions (excluding performed, and Figures 3 and 4 give the adsorption isotherms of DMAEMA from the copolymerization mixture) also allows to NIP and UA-MIP-7-16 for UA and xanthine. improve biogenic properties of sorbents [26]. Thus, UA was The isotherms in Figure 3 (a,b) show sorption of UA by dissolved in ethylenediamine (7 mol%) and diethylamine UA-MIP-7-16 and NIP at different temperatures. The sorption (16 mol%), and UA-MIP-5 and UA-MIP-6, were synthesized of UA from model aqueous solution (with and without 0.9 % respectively. These sorbents were based on EGDMA. The UA- of NaCl) was studied in the wide range of UA concentrations. MIP-5 sample can absorb UA only from the model aqueous The sorption capacity of UA-MIP-7-16 considerably exceeded solution, but UA-MIP-6 can absorb UA both from aqueous the sorption capacity of NIP, especially when UA concentration solution (160 mmol/g) and peritoneal liquid (40 mmol/g). was higher than 300 µmol/L. UA was able to interact with UA- UA-MIP-7-16 was also synthesized with addition of UA MIP-7-16 and demonstrated both non-specific and specific (16 mol%) dissolved in diethylamine. UA-MIP-7-16 was binding (sorption in imprinted cavities). The character of UA obtained in the form of spherical granules (400-200 μm). sorption isotherms had a similar tendency in the case of UA- UA-MIP-7-16 can absorb UA both from model aqueous MIP-7-16 and NIP (Figure 3b). solution (180 mmol/g) and peritoneal liquid (70 mmol/g) and In the studies of sorption specificity, we also used the demonstrates higher sorption capacity than other MIPs. That is closest structural analogue of UA (xanthine) and model why this sorbent was selected for further studies of specificity aqueous solutions with and without 0.9 % of NaCl. Xanthine of UA sorption. (2,6-dihydroxypurine) is a precursor of UA in the purine

21 A.P. Leshchinskaya et al.

biosynthesis. The xanthine sorption capacity of UA-MIP-7-16 3.3. Effect of temperature on sorption equilibrium and NIP does not change over the wide range of concentrations. To study the nature of intermolecular interactions between UA Moreover, the xanthine sorption capacity of NIP was higher than and complementary cavities, we need to understand the effects that of UA-MIP-7-16. Therefore, both xanthine and UA take part in of temperature on the binding characteristics of UA-MIP-7-16. the same non-specific interactions with polymeric sorbent matrix. Table 3 shows the effect of temperature on sorption of UA and To understand the nature of intermolecular interactions, the xanthine by NIP and UA-MIP-7-16. main thermodynamic functions were calculated using the model The sorption of UA by NIP from model solutions and solutions of UA and xanthine. peritoneal liquid is an endothermic enthalpy-controlled a b

Figure 4. Equilibrium sorption of xanthine from water solution by UA-MIP-7-16 and NIP with 0.9% NaCl and without NaCl.

Table 3. Distribution coefficients and thermodynamic functions of sorption UA andxanthine by NIP and UA-MIP-7-16.

293 К 310 К

∆G ∆H T∆S ∆G ∆H T∆S Γ Γ 1 kJ/mol kJ/mol kJ/mol 2 kJ/mol kJ/mol kJ/mol

URIC ACID

water solution of uric acid without NaCl

NIP 37.3 - 8.8 30.2 38.9 73.4 - 10.4 30.2 40.6

UA-MIP-7-16 72.4 - 10.4 - 18.2 - 7.8 47.8 - 9.9 - 18 - 8.2

water solution of uric acid with 0.9 % NaCL

NIP 15.6 - 6.7 108.7 115.4 187.9 - 12.7 108.7 121.5

UA-MIP-7-16 36.5 - 8.7 176.2 184.9 1940.4 - 18.4 176.2 194.6

peritoneal liquid

NIP - - - - 10 - 5.9 102.1 108

UA-MIP-7-16 30 - 8.3 - 62.6 - 54.3 7.3 - 5.1 - 62.6 - 57.4

XANTHINE

water solution of xanthine without NaCl

NIP 39.68 - 8.9 - 28.3 -19.3 21 - 7.8 -28.3 - 20.5

UA-MIP-7-16 1.43 -2.05 -8.6 -6.6 12 - 6.4 45.3 51.6

water solution of xanthine with 0.9 % NaCL

NIP 13.67 -3.4 22.7 29.1 22.9 - 8.1 22.7 30.8

UA-MIP-7-16 4. 35 - 3.6 45.3 48.8 11.5 - 6.3 108.4 114.7

22 Selective sorption of uric acid by novel molecularly imprinted polymers

process, in which non-specific interactions between UA and 3.5. Sorption dynamics of uric acid non-imprinting NIP matrix are realized. The dynamics of UA sorption from model solutions and serum by The sorption of UA by UA-MIP-7-16 from aqueous UA-MIP-7-16 was studied (Figure 7). Understanding dynamics solution without NaCl and peritoneal liquid is an exothermic of UA desorption is important for developing plasmasorption enthalpy-controlled process. Since UA-MIP-7-16 possesses method of sorbent regeneration. Desorption of UA was specific complementary cavities, this fact determines the performed with the aid of the mixture of 0.05 N HCl with 80% nature of molecular interactions; in the first place, they are of isopropanol. The relative masses of the adsorbed UA were hydrogen bonds between UA and sorbent [27]. Moreover, this calculated (Table 5). fact can be tested experimentally. The UA sorption equilibrium The most selective dynamic sorption was reached on experiments were also carried out with addition of urea (0.1 the column with parameters D×H = 10×80 mm. This column N solution), and in this case sorption was suppressed. In a addition, the formation of the complex in the first stage of UA- MIP-7-16 synthesis takes place at the expense of hydrogen bonds between UA template molecules and the monomer (EGDMA). The main thermodynamic functions were also calculated for the processes of xanthine sorption by NIP and UA- MIP-7-16. Comparison between thermodynamic functions of xanthine and UA sorption shows the difference in mechanisms of binding between these small molecules and the synthesized sorbents. This fact confirms the presence of the cavities specific for UA in the UA-MIP-7-16 polymer.

3.4. Kinetics of uric acid sorption The study of sorption kinetics allowed us to determine the b mechanism of diffusion of sorbate into sorbent phase and the influence of sorption medium and physicochemical factors on the diffusion flow. The experimental data were interpreted using kinetic curves F=f( t ), (Figure 5 (a,b,c)). These curves showed dependence between degree of saturation of sorbent with sorbate and sorption time. The main kinetic parameters (diffusion coefficient ( D , cm2×s-1) and average sorption time ( t, ) were necessary for prediction of the UA sorption behavior in dynamic conditions. The kinetic studies of sorption of UA by UA-MIP-7-16 without NaCl have demonstrated that mass transfer proceeds according to the intraparticle diffusion mechanism (Figure 5a). The kinetic parameters varied little with UA-MIP-7-16 particle size. The c improvement of UA sorption kinetics in imprinted matrix of UA-MIP-7-16 was observed after addition of NaCl (0.9 %) serving as a source of competing ions (Figure 5b). Hence, mass transfer parameters show weak dependence on the UA-MIP-7-16 particle size. The straight segments of curves in Figure 5c (F=0.01÷0.4) also confirm that UA sorption from peritoneal liquid proceeds according to the intraparticle diffusion mechanism. The main kinetic parameters are presented in Table 4. The diffusion coefficients and average sorption times were calculated using both the Boyd model and the “shall and core” model. The dimensions of sorption layer (L) of UA- MIP-7-16 were measured experimentally. The sorption layer L has a thickness of 15 μm (Figure 6). The similar orders of diffusion coefficients (Table 3) indicate that the cavities Figure 5. Sorption kinetics of UA by UA-MIP-7-16. a - sorption of UA from water solution without NaCl; b - sorption of UA from water complementary to UA are located mainly on the surface of solution with 0.9% NaCl; c - sorption of UA from peritoneal

UA-MIP-7-16. liquid. Dp is the diameter of UA-MIP-7-16 particles.

23 A.P. Leshchinskaya et al.

Table 4. The main kinetic parameters of UA sorption by UA-MIP-7-16.

Particles diameter of model Boyd «shell and core» tga UA-MIP-7-16, mcrm t, min D× 1011 , cm2×s-1 t, min D× 1011 , cm2×s-1 water solution of uric acid without NaCl

71-100 0.007 406 9.1 275.4 5.4

100-160 0.006 193 14 131 9.9

160-315 0.013 123 15.8 86 13.7

315-400 0.03 276 13.5 193 6.5

water solution of uric acid with 0.9 % NaCL

71-100 0.009 146 7.9 87 10.1

100-160 0.009 193 6.9 135 8.2

160-315 0.002 99 16.7 77 17

315-400 0.002 96 15.9 65 17.8

peritoneal liquid

71-100 0.003 160 8.5 112 9.3

100-160 0.003 400 4.5 272 4.0

160-315 0.002 410 5.5 278 4.1

315-400 0.003 169 9.8 118 10.1

was used in the experiments on UA sorption from serum (UA concentration was 0.509 μmol/L). To study the UA-MIP-7-16 affinity, the concentrations of the main serum components (UA, albumin, glucose, cholesterol, creatinine, urea) were measured. Decrease (%) in UA level and levels of the main components of serum was calculated (Figure 8). Percentage of sorption by UA- MIP-7-16 (%) for UA was higher than those for other substances. This fact demonstrated high selectivity of UA-MIP-7-16.

4. Conclusions

In our work, uric acid-imprinted polymers based on DMAEMA and EGDMA have been synthesized successfully and used for selective UA sorption from model solutions and serum. Figure 6. Equilibrium sorption of UA from water solution by UA-MIP-7-16 The UA-MIP-7-16 polymer (EGDMA-crosslinked system) which particles of different diameters. was synthesized with UA templates (16 mol%) was introduced into polymerization mixture and chosen as the best MIP. The study of UA and xanthine equilibrium sorption and demonstrated by biochemical analysis of serum containing thermodynamic functions using UA-MIP-7-16 and NIP UA and other components before and after sorption by this (reference non-imprinted polymeric sorbent) demonstrated polymer. Therefore, it can be expected that the UA-imprinted the prevalence of specific binding of UA by the optimal novel sorbent will be a promising material in blood purification. The imprinted sorbent. results obtained in this work were used for development of The study of the main kinetic parameters of UA sorption blood purification materials with high recognition selectivity. by UA-MIP-7-16 from aqueous solutions and peritoneal liquid demonstrated the even distribution of specific binding sites Acknowledgements and their high accessibility in the structurally heterogeneous imprinted matrix. The effective diffusion coefficient and The authors are grateful to the Federal State Budgetary Institute distribution constant of UA sorption from the peritoneal liquid «The Nikiforov Russian Center of Emergency and Radiation lead to selective binding of UA with UA-MIP-7-16 in dynamic Medicine», the Ministry of Russian Federation for Civil Defense, conditions. The selectivity of UA-MIP-7-16 was further Emergencies and Elimination of Consequences of Natural Disasters

24 Selective sorption of uric acid by novel molecularly imprinted polymers

Table 5. Sorption dynamics of UA by UA-MIP-7-16 from peritoneal liquid.

Columns parameters D×H, mm Speed W, mL/min relative masses of UA adsorbed, %

1 D×H=10×80 0.2 15.3

2 D×H=10×80 0.1 18.9

3 D×H=10×80 0.4 19.6

4 D×H=20×22 0.2 13

5 D×H=20×60 0.2 17

Figure 7. Sorption dynamics of UA by UA-MIP-7-16 from peritoneal liquid.

Figure 8. Decrease (%) in concentrations of UA and other main components in serum.

25 A.P. Leshchinskaya et al.

(NRCERM, EMERCOM of Russia) and the Saint-Petersburg I. I. This work is supported by the Russian Foundation for Basic Mechnikov State Medical Academy for aid in research. Research (Grant № 09-03-00516-а).

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