Selective Sorption of Uric Acid by Novel Molecularly Imprinted Polymers
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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.