Preparation And Application Of Nicotinamide Imprinted Polymer Microspheres

Molecularly imprinted technology (MIT) is a new molecular recognition technology developed on the basis of interaction between antigens and antibodies, which exhibits extraordinary affinity to the target molecule. The remarkable selectivity of MIT derives from the molecularly imprinted polymers (MIP), a kind of synthetic polymers which is matched well with the target molecule by the spatial structure and binding sites. MIP known as "artificial antibodies", has pre-scheduled, high selectivity, stable performance, simple preparation and other characteristics, and widely be used in biomimetic sensors, solid phase extraction, antibody modeling, liquid stationary phase, enzyme catalysis simulation, chiral separation, drug delivery and other areas.Molecularly Imprinted-Solid Phase Extraction (MISPE) is a new separation technology associated MIT with Solid Phase Extraction (SPE), which used the MIP as SPE sorbents for separation, enrichment and analysis of a specific target molecule in complex samples. MISPE not only has the advantages of SPE, but also give the MSPE better function, such as selectively enriched target molecules in samples, effective removed interference components and impurities. MISPE is a powerful tool for separation and analysis of trace target molecule in complex matrix.In this paper, nicotinamide was used as the target molecule (template), NAM-MIP was synthesized by different polymerization methods, and the optimal method and synthetic parameters were determined. NAM-MIP was characterized and evaluated after preparing under the optimal conditions. Used NAM-MIP as SPE adsorbents, the special affinity and high selectivity of MISPE to the samples of NAM in serum, urine was investigated.1. Objectives and ContentsNAM-MIP microspeheres were prepared by two-step swelling polymerization with NAM as the template. Used NAM-MIP as SPE sorbents to prepare NAM-MISPE for separation and enrichment of NAM in serum or urine samples in order to established a pre-treatment methods for biological samples.(1) Preparation and process optimization of NAM-MIP(2) Structure characterization and property evaluation of NAM-MIP(3) Application of NAM-MISPE to the analysis of biological samples2. Methods2.1 Preparation and process optimization of NAM-MIP(1) Different NAM-MIP and NIP were prepared by swelling polymerization, bulk polymerization, suspension polymerization and precipitation polymerization respectively. Compare to the equilibrium adsorption capacity Q, specific adsorption⊿Q, distribution coefficient K and imprinting factor a of each MIP, the proper polymerization method was determined.(2) With the adsorption capacity Q and polymer morphology as the indicator, the process parameters influenced NAM-MIP preparation including the amount of MAA, porogen, stirring speed, Vwater/VEGDMA, dispersants and emulsifiers and polymerization temperature was investigated.2.2 Structure characterization and property evaluation of NAM-MIP(1) NAM-MIP synthesized under the optimal conditions was characterized by SEM, particle size analysis and IR spectroscopy.(2) The adsorption property of NAM-MIP toward NAM was evaluated by adsorption isotherm, Scatchard analysis and kinetic curve.(3) The binding property and completive adsorption of NAM-MIP towards different substrates was evaluated by static adsorption isotherm and MISPE analysis loaded with the substrate mixtures.2.3 Application of NAM-MISPE to the analysis of biological samplesThe urine and serum sample containing NAM was loaded on MISPE, after washing, eluting under the optimal condition, NAM was determined in the efflutes by HPLC.3. Results3.1 Determining NAM-MIP preparation method3.1 Determining preparation method of NAM-MIPThe adsorption capacity Q and specific adsorption⊿Q of NAM-MIP prepared by swelling polymerization was higher than that of MIP prepared by precipitation polymerization, bulk polymerization and suspension polymerization. Considered the polymer particle size, yield, adsorption capacity and specific adsorption, the two-step swelling polymerization was selected to prepare NAM-MIP, whose Q, KMIp and a was 38.2μmol/g、28.7 mL/g、2.96 respectively.3.2 Optimizing preparation process of NAM-MIPThe optimal parameters of two-step swelling polymerization were determined through the single factor investigation with the adsorption Q and particle shape as the index:lmmol NAM,6 mmol MAA,30 mmol EGDMA,10 mL toluene,75℃polymerization temperature,200 rpm stirring speed,0.15% SDS,1.75% PVA,25:1 Vwater/VEGDMA.The obtained NAM-MIP showed proper single-dispersion, uniform particle and good static adsorption and selective adsorption performance.3.3 Characterization the structure of NAM-MIPThe surface of NAM-MIP was roughness and had a complex porous structure, while NIP was smooth. The particle size was in the range of 10～40μm, the average was about 25μm. It showed that all EGDMA and MAA was participated in the polymerization reaction by IR. The obvious stretching vibration of hydroxyl group in 3630 cm-1 was observed in MIP before and after elution of the template molecule.3.4 Binding properties of NAM-MIP to NAMThe adsorption of NAM-MIP to NAM was significantly higher than that of NIP, the adsorption basically reached equilibrium at the concentration of 3 mmol/L NAM. Scatchard analysis showed that two different binding sites, the low affinity binding sites (Kd1=0.178 mmol/L, Qmaxi=15.26μmol/g) and high affinity binding site (Kd2=3.65 mmol/L, Qmax2=149.34μmol/g), were existed between NAM and NAM-MIP. And one equivalent binding site (Kd=2.06 mmol/L, Qmax=50.0μmol/g) was found between NAM and NIP. It was observed that the adsorption rate was rapid at the beginning of 1.5 h and reached equilibrium about 7h by kinetic curve.3.5 The binding properties and competitive adsorption of NAM NAM-MIP to structural analoguesNAM-MIP also showed some binding property to the structure analogues of NAM, the adsorption capacity of NA (Q=23.8μmol/g) was highest, followed by 3-PM (Q= 18.8μmol/g) and BA (Q=16.5μmol/g), BM (Q=6.3μmol/g) was the weakest. The mixture substrate sample was loaded on MISPE column, after washing, elution, it was deduced that the competitive ability of NAM to the binding sites of NAM-MIP was the strongest, and the recovery of NAM in elute was 88.3%, followed by NA (56.6%) and 3-PM (29.2%).3.6 NAM-MISPE used in the analysis of biological samplesThe optimal process of MISPE was determined, which the cartridge was washed with 0.5 mL of methanol, and eluted with 3.0 mL×2 of methanol/acetic acid (9:1, v /v). The standard curve equation of NAM in urine:y=96.472x-80.254 (r=0.9948); in serum:y=0.0145x+0.159 (r=0.9975), which in the range of 0.499μg/mL～19.96μg/mL of NAM in urine and serum, linear relationship was satisfactory, LOD was 0.3μg/mL, LOQ was 0.9μg/mL. The precision of RSD was lower than 2.86%. The relative recovery of NAM in urine was 98.8%～86.0%, RSD was 1.89%～4.44%; in serum was 96.8%～87.0%, RSD was 2.27%～3.19%.4. Conclusions4.1 The two-step swelling polymerization was feasible for the preparation of NAM-MIP microspheres. NAM-MIP synthesized under the optimal conditions was round and had proper single-dispersion, uniform particle and good static adsorption and selective adsorption performance.4.2 The adsorption equilibrium of NAM-MIP was related to the concentration of the substrate and adsorption time. There were two types of non-equivalent binding sites between NAM-MIP and NAM, that was the low affinity binding sites driven by non-specific adsorption, and the high affinity binding sites driven by specific adsorption. Only one class of equivalent non-specific binding sites was found between NIP and NAM.4.3 The adsorption of NAM-MIP to NAM was selectivity and specificity. The mechanism of the recognition was deduced that the hole of MIP was matched with the spatial structure of NAM, and hydroxyl group of MAA in NAM-MIP could interact with pyridine ring and carbonyl of NAM by hydrogen bonds.4.4 NAM-MISPE used NAM-MIP as the sorbents could specific separate and enrichment NAM in serum and urine. The established analysis method based on MISPE was specific, accurate, reliable and simple, and could meet the test requirements of biological samples.