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1、Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 288294Monodisperse, molecularly imprinted polymers for creatinine by modied precipitation polymerization and their applications to creatinine assays for human serum and urineChitose Miura, Noriko Funaya, Hisami Matsunaga, Jun HaginakaSchool

2、 of Pharmacy and Pharmaceutical Sciences, Mukogawa Womens University, 11-68, Koshien Kyuban-cho, Nishinomiya 663-8179, Japana r t i c l ei n f o a b s t r a c t Article history:Received 23 May 2013Received in revised form 27 July 2013 Accepted 29 July 2013Available online 7 August 2013Molecularly im

3、printed polymers (MIPs) for creatinine were prepared by modied precipitation polymer- ization using methacrylic acid as a functional monomer and divinylbenzene as a crosslinker. The prepared MIPs were monodispersed with a narrow particle size distribution. Binding experiments and Scatchardanalyses r

4、evealed that two classes of binding sites, high- and low-afsites, were formed on the MIPs.The retention and molecular-recognition properties of the MIPs were evaluated by hydrophilic interac- tion chromatography using a mixture of ammonium acetate buffer and acetonitrile as a mobile phase.Keywords:M

5、olecularly imprinted polymer Modied precipitation polymerization BioanalysisCreatinine CreatineWith an increase of acetonitrile content, the retention factor of creatinine was increased on the MIP. In addition to shape recognition, hydrophilic interactions seemed to enhance the recognition of creati

6、nine on the MIP. The MIPs molecular-recognition ability was specic for creatinine; the structurally related compounds such as hydantoin, 1-methylhydantoin, 2-pyrrolidone, N-hydroxysuccinimide and creatine were not recognized. Furthermore, the creatinine concentrations in human serum and urine were s

7、uc- cessfully determined by direct injection of the deproteinized serum and diluted urine samples onto the MIP.© 2013 Elsevier B.V.1. Introductionchromatography have been the primary separation methods for creatinine in biological uids. Recently, hydrophilic interaction chromatography (HILIC) 1

8、3,14 has shown promise as an assay for hydrophilic creatinine. Additionally, porous graphite 15 andCreatinine is a spontaneous, nonenzymatic degradation prod- uct of creatine or phosphocreatine in vertebrates 1. Creatinine levels are an important clinical index in assessing renal and mus- cular func

9、tions. The normal creatinine range in blood is 44106 M (5.814 g/mL). Levels over 140 M (16 g/mL) require clini- cal investigation, and levels above 530 M (60 g/mL) indicate severe renal impairment 2. The normal range for urine creati- nine is 2.523 mM (0.282.59 mg/mL) 3. Furthermore, creatininecatio

10、n-exchange 16 columns have proven useful for qufy-ing creatinine in biological uids. The LC detection methods in these creatinine assays have included UV absorption 12,14,16 and tandem MS 10,11,13,15. CE, CZE 4,1719 and micellar elec-trokinetic chromatography 20,21 combined with UV absorption 4,17,1

11、9,20, conductivity 18 or electrochemical 21 detection could be utilized for creatinine assays in serum and urine.Molecular imprinting techniques are attractive because specic recognition sites for a target molecule can be easily molded in syn- thetic polymer networks 2224. Prepared MIPs have been ut

12、ilizedconcentration is used as a standardization tool for qu apeutic drugs, illicit drugs and xenobiotics in urine 4.fying ther-Enzymatic and Jaffé reaction-based spectroscopic methods havebeen used extensively for creatinine qu ids 1,5. However, neither method iscation in biological u- from in

13、terferences 1.as chromatographic media, sensors, articialbodies and cata-The use of sequential enzymes permits highly selective enzymaticlysts 2224. MIPs for creatinine have been prepared by several methods: bulk polymerization 25,26, solgel 27 and surfacequcation; however, the method is expensive 6

14、8. Alterna-tive sensitive or selective approaches include separation methodsimprinting 28 techniques. Recently, we prepared a monodis- perse MIP for creatinine by modied precipitation polymerization 29. Because creatinine is hydrophilic, it does not dissolve in porogenic solvents. Therefore, creatin

15、ine was rst dissolved in a small portion of water, and then a MIP for creatinine was pre- pared by precipitation polymerization. The use of water to dissolve creatinine distinguished this method from other precipitation poly- merizations. Furthermore, MIPs for creatinine have been appliedsuch as LC,

16、 GC and capillary electrophoresis (CE) and biosensors based on immobilized enzymes or molecularly imprinted poly- mers (MIPs) 8,9. In LC, reversed-phase 10,11 and ion-pair 12 Corresponding author. Tel.:45 9949; fax:41 2792.address: haginakamukogawa-u.ac.jp (J. Haginaka).0731-7085/$ see front matter

17、© 2013 Elsevier B.V.Contents lists available at ScienceDirectJournal of Pharmaceutical and Biomedical Analysisjournal homepage: C. Miura et al. / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 288294289to real samples: biosensors based on MIPs for creatinine were used for assays of

18、 creatinine in serum and urine 30,31. Lastly, MIP/quantum dot composite nanoparticles have been prepared for detecting creatinine in urine 32.In this study, monodisperse MIPs for creatinine were prepared by modied precipitation polymerization. The binding properties of the prepared MIPs were examine

19、d by binding experiments and Scatchard analyses. The MIPs retention and molecular-recognition properties were evaluated by HILIC using a mixture of ammonium acetate buffer and acetonitrile as a mobile phase. In addition, the2.4. Scanning electron micrographsScanning electron micrographs (SEMs) were

20、obtained using a Mighty-8 instrument (Technex, Machida, Japan).2.5. Chromatographic evaluation of the MIPsTo evaluate their chromatographic characteristics, the obtained polymers were slurried in methanol-2-propanol (2/1, v/v) and packed into stainless-steel columns using methanol at a constant pres

21、sure of 9.8 Mpa. The LC system was composed of a PU-980 pump and a UV-970 spectrophotometer from Jasco (Tokyo, Japan), a C-R6A integrator (Shimadzu, Kyoto, Japan), a Rheodyne 7125 injec- tor with a 20- L loop (Rheodyne, Cotati, CA, USA) and a CO-1093C column oven (Uniows, Tokyo, Japan). The followin

22、g LC conditions were employed: a MIMIP for creatinine was used to qufy creatinine in human serumand urine by direct injection of the deproteinized serum and diluted urine samples onto the MIP.IP column (50 mm × 2.0 mm I.D.), a2. Experimental25 C column temperature, 2 mM ammonium acetate buffer

23、(pH7.0)acetonitrile (10/90, v/v) mobile phase, a 0.2 mL/min ow rate and detection at 210 nm.The retention factor (k) was calculated using the equation2.1. MaterialsDivinylbenzene (DVB) as a mixture of the 1,3- and 1,4-isomers, methacrylic acid (MAA) and 2,2 -azobis(isobutyronitrile) (AIBN) were purc

24、hased from Wako (Osaka, Japan). Creatinine, hydan- toin, 1-methylhydantoin, 2-pyrrolidone, N-hydroxysuccinimide and creatine were purchased from Nacalai Tesque (Kyoto, Japan). Control human serum (Seiken Liquid Normal V Plus) was purchased from Denka Seiken (Tokyo, Japan). The reagents and analytica

25、lgrade solvents were used without further purication. Puried water from a PURELAB® Ultra system (Organo, Tokyo, Japan) wasused in preparing mobile phases and sample solutions. Creatinine and its structurally related compounds are shown in Fig. 1.k = (tR t0)/t0, in which tR and t0 are the retent

26、ion times of theretained and unretained solutes, respectively. The retention time of the unretained solute, t0, was measured by injecting a solution with a water content that was slightly different from the mobile phase. The imprinting factor (IF) was calculated using the equation IF = kMIP/kNIP, in

27、 which kMIP and kNIP are the solute retention factors on an MIP column and its corresponding NIP column, respectively.2.6. Creatinine binding and Scatchard analysisA 10 mg sample of MIP3 or NIP3 was added to 1 mL acetonitrilewater (50/50, v/v) solutions containing 0.10.7 mmol/L creatinine. After sha

28、king the suspension at 25 C for 6 h, an aliquot of each sample was taken and ltered through2.2. MIP preparations by modied precipitation polymerizationa 0.45- m membrane lter. Thecreatinine concentration,MIPs for creatinine and non-imprinted polymers (NIPs) were prepared by a modied precipitation po

29、lymerization method as previously reported 29. Creatinine solutions were prepared by dissolving 1.5 mmol creatinine in 0.5, 0.75 and 1.0 mL of water. Each of these solutions was dissolved with the MAA functional monomer (6 mmol), the DVB crosslinker (28.8 mmol) and the AIBN initiator (1.9 mmol) in 1

30、28 mL of acetonitriletoluene (3/1, v/v). The resulting solutions were degassed with argon gas for 15 min and rotated slowly at 16 rpm using a Shellspin VS-60 rotor (Taitec, Tokyo, Japan). Afterward, the reaction temperature was increasedfrom 25 to 60 C over 2 h and held at 60 C for another 248 h.The

31、 prepared MIPs were labeled MIP1, MIP2 and MIP3, respec- tively. NIPs (NIP1, NIP2 and NIP3) were prepared similarly but without a template. After polymerization, the dispersed polymers were poured into 200 mL of methanol, and the supernatants were discarded after sedimentation. The polymers were red

32、ispersed in methanol, and the sedimentation procedure was repeated three times in methanol, once in water and twice in tetrahydrofuran. The m-sized polymer particles were collected using a membrane lter, washed with tetrahydrofuran and dried at room temperature.Creatinine, in the supernatant was det

33、ermined under the LC con- ditions described in Section 2.5, except a 2 mM ammonium acetatebuffer (pH 7.0)acetonitrile (5/95, v/v) mobile phase was used. The amount of creatinine bound to MIP3 or NIP3, Q, was determined by subtracting the Creatinine from the initial creatinine concen-tration. The dis

34、sociation constant (Kd) and the apparentumnumber of binding sites (Qmax) were determined by the Scatchard plot using the equation, Q/Creatinine= (Qmax Q)/Kd.2.7. Method validation and qu serum and urine samplescation of creatinine in humanThe employed LC system consisted of a LC-10ADvp pump, a SPD-

35、10Avp spectrophotometer, a C-R6A integrator (all from Shimadzu, Kyoto, Japan), a Rheodyne 7125 injector with a 20- L loop (Rheo- dyne, Cotati, CA, USA) and a CO-1093C column oven (Uniows, Tokyo, Japan).The serum samples were spiked with six different creatinine amounts to create nal creatinine conce

36、ntrations of 0.5, 1, 2, 3, 4 and 5 g/mL. A 500- L aliquot of each serum sample was deproteinizedwith 2 mL of acetonitrile and then centrifuged at 10,000 × g for5 min. For analysis, a 5- L aliquot of each supernatant was loaded2.3. Porosity measurementsinto an LC system with a MIP3 column (50 mm

37、 × 2.0 mm I.D.). Thefollowing LC conditions were employed: a 25 C column tempera- ture, a 2 mM ammonium acetate buffer (pH 7.0)acetonitrile (7/93, v/v) mobile phase, a 0.2 mL/min ow rate and detection at 235 nm. Urine was collected from a healthy volunteer. The urine samples were spiked with ve

38、 different creatinine amounts to create nal creatinine concentrations of 0.1, 0.2, 0.5, 1.0 and 2.0 mg/mL. A 100- L aliquot of each urine sample was diluted with 2 mL of ace- tonitrile and centrifuged at 10,000 × g for 5 min. For analysis, a 5- LThe surface areas and porosities of the MIPs and

39、NIPs were mea- sured by nitrogen adsorption porosimetry using a TriStar surface area and porosity analyzer (Micrometrics Instruments, Norcross, GA, USA). Prior to measurement, 200 mg samples of the polymersweeated at 80 C for 5 h in vacuo. The specic surface areas werecalculated using the BET method

40、, and the specic pore volumes and average pore diameters were calculated by the BJH method.290C. Miura et al. / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 288294Fig. 1. Creatinine and the structurally related compounds used in this study.aliquot of each supernatant was loaded into a

41、n LC system under the conditions employed for the serum samples, except a 2 mM ammo- nium acetate buffer (pH 7.0)acetonitrile (8/92, v/v) mobile phase was used.The calibration graphs for human serum and urine weremodied precipitation polymerizationprocedure affordedmonodisperse MIPs for creatinine.

42、Table 1 also lists the specicsurface areas, specic pore volumes and average pore diameters of MIPs and NIPs. The synthesized MIPs and NIPs had high specic surface areas resulting from their microporous structures, and the MIPs and NIPs possessed similar morphologies. Furthermore, the specic water vo

43、lume used in preparation did not affect the morphology.constructed by plotting the creatinine peak heightthecreatinine concentration. The intra- and inter-day precision and accuracy data were obtained from the spiked serum and urine sam- ple assays.3.2. Evaluation of the MIPs for creatinine3. Result

44、s and discussionThe MIPs were rst evaluated in reversed-phase chromatogra- phy using a mixture of sodium phosphate buffer and acetonitrile as a mobile phase. Under these conditions, creatinine was not retained on the MIPs. The MIPs were then evaluated in HILIC using a mix- ture of 2 mM ammonium acet

45、ate buffer (pH 7.0) and acetonitrile as a mobile phase. These conditions resulted in the retention of creatinine.Fig. 3 shows the effect of acetonitrile content on the retention factors of creatinine on MIP3 and NIP3. With an increase of acetoni- trile content, the retention factors of creatinine on

46、 the MIP3 and3.1. Preparation of the MIPs for creatinineThree different MIPs for creatinine were prepared by changing the water volume used to dissolve the xed amount of creatinine template. The synthesis of MIP1, MIP2 and MIP3 used 0.5, 0.75 and1.0 mL of water, respectively. Fig. 2af shows the SEMs

47、 of MIP1, MIP2, MIP3 and the corresponding NIPs, respectively. The MIP and NIP particle diameters are listed in Table 1. Size uniformity was observed for the individual MIPs and NIPs, indicating that theC. Miura et al. / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 288294291Fig. 2. Sc

48、anning electron micrographs of (a) MIP1, (b) MIP2, (c) MIP3, (d) NIP1, (e) NIP2 and (f) NIP3.Table 1Physical properties of MIPs and NIPs.PolymerSpecic surface area (m2 /g)Specic pore volume (cm3 /g)Average pore diameter (nm)Average particle diameter ( m)MIP1 MIP2 MIP3 NIP1 NIP2 NIP3528495497543a 458

49、0.190.170.200.26 0.183.03.03.24.13.13.08 ± 0.093.53 ± 0.023.91 ± 0.054.00 ± 0.044.86 ± 0.064.34 ± 0.05a Not measured.NIP3 were increased. MIP3 afforded larger retention factors. These results indicated that in addition to shape recognition, hydrophilic interactions betw

50、een creatinine and the MIP enhanced creatinine retention. Although the retention and imprinting factors of creat- inine on MIP1, MIP2 and MIP3 were similar, the imprinting factor on MIP3 was slightly higher. These results revealed that the water volume used in the MIP preparation affected neither th

51、eir reten- tion nor molecular-recognition properties, and the results further supported that their morphologies were not affected by the watervolume employed. Therefore, in the subsequent experiments, MIP3 and NIP3 were used.The retention and molecular-recognition properties of struc- turally relate

52、d compounds such as hydantoin, 1-methylhydantoin, 2-pyrrolidone, N-hydroxysuccinimide and creatine were investi- gated. Fig. 4 compares the imprinting factors of these compoundsFig. 3. Effects of acetonitrile content on the retention factor of creatinine onFig. 4. Imprinting factors of creatinine, h

53、ydantoin, 1-methylhydantoin, 2- pyrrolidone, N-hydroxysuccinimide and creatine. LC conditions as in Fig. 3 except a 2 mM ammonium acetate buffer (pH 7.0)acetonitrile (10/90, v/v) mobile phase was employed.MIP3 and NIP3. LC conditions: 50 mm × 2.0 mm I.D. column size, 25 C columntemperature, 2 m

54、M ammonium acetate buffer (pH 7.0)acetonitrile mobile phase,0.2 mL/min ow rate, 210 nm detection and 500 ng sample sizes.292C. Miura et al. / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 288294Fig. 5. Binding isotherms of creatinine to MIP3 and NIP3.with the creatinine imprinting fact

55、or. For that experiment, a 2 mM ammonium acetate buffer (pH 7.0)acetonitrile (10/90, v/v) mobile phase was used. The imprinting factors of creatinine, 2-pyrrolidone, N-hydroxysuccinimide and creatine were 3.6, 0.94, 1.1, and 0.86, respectively. Because hydantoin and 1-methylhydantoin were not retain

56、ed on either MIP3 or NIP3, their imprinting factors could not be calculated accurately. These results indicated that the MIP rec- ognized creatinine and did not recognize the structurally related compounds.Fig. 7. Chromatograms of human serum samples on (a) NIP3 and (b) MIP3. LC con-ditions: 50 mm &

57、#215; 2.0 mm I.D. column, 25 C column temperature, 2 mM ammoniumacetate buffer (pH 7.0)acetonitrile (7/93, v/v) mobile phase, 0.2 mL/min ow rate, 235 nm detection and 5 L injection volume. The creatinine concentration was esti- mated to be 11 g/mL.The creatinine binding isotherms in these experiment

58、s are notable when compared with the previous results of others. For a MIP prepared using the polymerizable Lewis acidic zinc(II)cylclen complex and ethylene glycol dimethacrylate, the creatinine binding3.3. Binding experiments of creatinine to the MIP and NIPisotherm tted well to a one-site binding mwith Kd and Qmaxvalues of 2.5 × 105 mol/L and 2.9