Improving the Identification of Lysine-Acetylated Peptides Using a Molecularly Imprinted Monolith Prepared by a Deep Eutectic Solvent Monomer.

To overcome the identification challenge of low-abundance lysine acetylation (Kac), a novel approach based on a molecularly imprinted polymer (MIP) was developed to improve the extraction capacity of Kac peptides in real samples. Green deep eutectic solvents (DESs) were introduced and used as one of the synergistic functional monomers with zinc acrylate (ZnA). Glycine-glycine-alanine-lysine(ac)-arginine (GGAKacR) was chosen as a template and N,N'-methylenbisacrylamide (MBAA) was used as a cross-linker. The obtained GGAKacR-MIP had excellent selectivity for the template with an imprinting factor (IF) of up to 21.4. The histone digest addition experiment demonstrated that GGAKacR-MIP could successfully extract GGAKacR from a complex sample. Finally, the application to the extraction of Kac peptides from mouse liver protein digestion was studied in detail. The number of Kac peptides and Kac proteins identified was 130 and 110, which were 3.71-fold and 3.93-fold higher than those of the untreated sample. In addition, the number of peptides and proteins identified after treatment increased from 5535 and 1092 to 17 149 and 4037 (3.10-fold and 3.70-fold, respectively). The results showed that the obtained MIP may provide an effective technical tool for the identification of Kac-modification and peptide fractionation, as well as a potential approach for simultaneously identifying post-translational-modified proteomic and proteomic information.

Improving sorption performance of a molecularly imprinted monolithic column by doping mesoporous molecular sieve SBA-15.

For the first time a hybrid molecularly imprinted polymer (MIP) doped with 3-(trimethoxysilyl) propyl methacrylate (γ-MPS)-modified mesoporous molecular sieve SBA-15 for target peptide recognition has been developed. Zinc acrylate and methacrylic acid were used as binary functional monomers, and ethylene dimethacrylate was used as cross-linking agent to prepare an imprinted monolith against Val-Tyr-Ala-Leu-Lys(glutarylation) (VYALKglu). The morphology of the polymers was characterized by scanning electron microscopy, FT-IR spectroscopy, energy dispersive spectroscopy, and 1H NMR. The SBA-15-MPS MIP showed high recovery of 87.1% and the IF of 12.9 for the enrichment of the template peptide. When the template peptide concentration ranged from 5 to 90 µg mL-1, the correlation coefficients (R2) for the calibration function obtained was better 0.999. The limit of detection (LOD, 0.30 µg mL-1) and limit of quantification (LOQ, 1.0 µg mL-1) were achieved for signal-to-noise ratios of 3:1 and 10:1, respectively. When other kinds of synthetic peptides were used as analogs, the selectivity of the SBA-15-MPS MIP was much better than the SBA-15-MPS NIP (without template peptides) with relative selectivity coefficients of 52.8-265. In contrast, little quinolones and biogenic amines are adsorbed with the SBA-15-MPS MIP. The SBA-15-MPS MIP could enrich VYALKglu from spiked histone digestion with the average recovery of 87.8% and the relative standard deviation (RSD) of 0.99%. As a conclusion, doping of SBA-15 is an effective approach to the improvement of performance of molecularly imprinted monolith.

Cost-effective imprinting to minimize consumption of template in room-temperature ionic liquid for fast purification of chlorogenic acid from the extract of E. ulmoides leaves.

One of the main challenges in large-scale applications of molecularly imprinted polymers (MIPs) is the significant amount of template needed in polymer preparation. A new strategy based on room-temperature ionic liquids (RTILs) was suggested to solve this problem by reducing the amount of template in the polymerization recipe. The MIP was synthesized with a mixture of dimethyl sulfoxide and RTIL (1-butyl-3-methylimidazolium tetrafluoroborate) as porogen, in which chlorogenic acid (CGA) was used as template, 4-vinylpyridine (4-VP) as functional monomer, and ethylene glycol dimethacrylate (EDMA) as cross-linker. The influence of polymerization variables, including CGA concentrations, and the ratio of 4-VP to EDMA on imprinting effect were investigated comprehensively. Moreover, the properties involving the column permeability, the number of binding sites, and the polymer morphology of the CGA-MIP monoliths were studied thoroughly. The MIP monolith had an excellent column permeability (1.53 × 10-13 m2) and allowed an ultra-fast on-line SPE, which dramatically shortens the separation time (< 10 min) and improves the separation efficiency. At high flow velocity (5.0 mL min-1), 50 µL of the extract from Eucommia ulmoides leaves can be loaded directly on the CGA-MIP monoliths and CGA with high purity can be obtained with a recovery of 89.01 ± 0.05%. As a conclusion, the resulting RTIL-induced approach of preparing MIP may be an effective tool in fabricating MIP in a low-cost way. Graphical abstract ᅟ.

Green Synthesis of Carbon Nanotubes-Reinforced Molecularly Imprinted Polymer Composites for Drug Delivery of Fenbufen.

The facile fabrication of single-walled carbon nanotubes (SWCNTs)-doping molecularly imprinted polymer (MIP) nanocomposite-based binary green porogen system, room-temperature ionic liquids (RTILs), and deep eutectic solvents (DESs) was developed for drug delivery system. With fenbufen (FB) as template molecule, 4-vinylpyridine (4-VP) was used as functional monomer, ethylene glycol dimethacrylate as cross-linking monomer, and 1-butyl-3-methylimidazoliumtetrafluoroborate and choline chloride/ethylene glycol as binary green solvent, in the presence of SWCNTs. The imprinting effect of the SWCNT-MIP composites was optimized by regulation of the amount of SWCNTs, ratio of RTILs and DES, and the composition of DES. Blue shifts of UV bands strongly suggested that interaction between 4-VP and FB can be enhanced due to SWCNT doping in the process of self-assembly. The reinforced imprinted effect of CNT-doping MIP can provide superior controlled release characteristics. Compared with the control MIP prepared without SWCNTs, the imprinting factor of the SWCNT-MIP composites exhibited a twofold increase. In the analysis for the FB release kinetics from all samples, the SWCNT-reinforced MIP produced the lowest value of drug diffusivity. The relative bioavailability of the SWCNT-MIP composites (F %) displayed the highest value of 143.3% compared with the commercial FB tablet, whereas the control MIP and SWCNT-non-MIP composites was only 48.3% and 44.4%, respectively. The results indicated that the SWCNT-MIP nanocomposites developed here have potentials as the controlled-release device.

Cost-effective imprinting combining macromolecular crowding and a dummy template for the fast purification of punicalagin from pomegranate husk extract.

The combination of molecular crowding and virtual imprinting was employed to develop a cost-effective method to prepare molecularly imprinted polymers. By using linear polymer polystyrene as a macromolecular crowding agent, an imprinted polymer recognizable to punicalagin had been successfully synthesized with punicalin as the dummy template. The resulting punicalin-imprinted polymer presented a remarkable selectivity to punicalagin with an imprinting factor of 3.17 even at extremely low consumption of the template (template/monomer ratio of 1:782). In contrast, the imprinted polymer synthesized without crowding agent, did not show any imprinting effect at so low template amount. The imprinted polymers made by combination of molecular crowding and virtual imprinting can be utilized for the fast separation of punicalagin from pomegranate husk extract after optimizing the protocol of solid-phase extraction with the recovery of 85.3 ± 1.2%.

Current trends in the development of molecularly imprinted polymers in CEC.

This review focused on the developments in the field of molecularly imprinted polymers (MIPs) for CEC since 2009. New preparation techniques of MIP-based CEC, such as, portable microchip with macroporous monolithic imprinted microchannel, and low cross-linking MIPs based on liquid crystalline monomers, were discussed. Using selected cases rather than a comprehensive review of the entire field, our goal is to highlight the studies of the interest with an emphasis on recent work, and offers suggestions for future development in the field of imprinted materials for CEC separation.

Molecular crowding-based imprinted monolithic column for capillary electrochromatography.

Molecular crowding is a new approach to stabilizing binding sites and improving molecular recognition. In this work, the concept was applied to the preparation of imprinted monolithic columns for CEC. The imprinted monolithic column was synthesized using a mixture of d-zopiclone (d-ZOP)(template), methacrylic acid, ethylene glycol dimethacrylate, and poly(methyl methacrylate) (PMMA) (molecular crowding agent). The resulting PMMA-based imprinted capillary was able to separate ZOP enantiomers in CEC mode. The resolution of enantiomer separation achieved on the d-ZOP-imprinted monolithic column was up to 2.09. Some polymerization factors, such as template-monomer molar ratio, functional monomer-cross-linker molar ratio and the composition of the porogen, on the imprinting effect of resulting molecularly imprinted polymer (MIP) monolithic column were systematically investigated. Chromatographic parameters, including pH values, the content of acetonitrile and the salt concentration on chiral separation were also studied. The results indicated the addition of PMMA resulted in MIPs with superior retention properties and excellent selectivity for d-ZOP, as compared to the MIPs prepared without addition of the crowding-inducing agent. The results revealed that molecular crowding is an effective method for the preparation of a highly efficient MIP stationary phase for chiral separation in CEC.

Macromolecular crowding-assisted fabrication of liquid-crystalline imprinted polymers.

A macromolecular crowding-assisted liquid-crystalline molecularly imprinted monolith (LC-MIM) was prepared successfully for the first time. The imprinted stationary phase was synthesized with polymethyl methacrylate (PMMA) or polystyrene (PS) as the crowding agent, 4-cyanophenyl dicyclohexyl propylene (CPCE) as the liquid-crystal monomer, and hydroquinidine as the pseudo-template for the chiral separation of cinchona alkaloids in HPLC. A low level of cross-linker (26%) has been found to be sufficient to achieve molecular recognition on the crowding-assisted LC-MIM due to the physical cross-linking of mesogenic groups in place of chemical cross-linking, and baseline separation of quinidine and quinine could be achieved with good resolution (R(s) = 2.96), selectivity factor (α = 2.16), and column efficiency (N = 2650 plates/m). In contrast, the LC-MIM prepared without crowding agents displayed the smallest diastereoselectivity (α = 1.90), while the crowding-assisted MIM with high level of cross-linker (80%) obtained the greatest selectivity factor (α = 7.65), but the lowest column efficiency (N = 177 plates/m).

Comparison of multi-recognition molecularly imprinted polymers for recognition of melamine, cyromazine, triamterene, and trimethoprim.

Three fragmental templates, including 2,4-diamino-6-methyl-1,3,5-triazine (DMT), cyromazine (CYR), and trimethoprim (TME), were used to prepare the fragment molecularly imprinted polymers (FMIPs), respectively, in polar ternary porogen which was composed of ionic liquid ([BMIM]BF4), methanol, and water. The morphology, specific surface areas, and selectivity of the obtained FMIPs for fragmental analogues were systematically characterized. The experimental results showed that the FMIPs possessed the best specific recognition ability to the relative template and the greatest imprinting factor (IF) was 5.25, 6.69, and 7.11 of DMT on DMT-MIPs, CYR on CYR-MIPs, and TME on TME-MIPs, respectively. In addition, DMT-MIPs also showed excellent recognition capability for fragmental analogues including CYR, melamine (MEL), triamterene (TAT), and TME, and the IFs were 2.08, 3.89, 2.18, and 2.60, respectively. The effects of pH and temperature on the retention of the fragmental and structural analogues were studied in detail. Van't Hoff analysis indicated that the retention and selectivity on FMIPs were an entropy-driven process, i.e., steric interaction. The resulting DMT-MIPs were used as a solid-phase extraction material to enrich CYR, MEL, TAT, and TME in different bio-matrix samples for high-performance liquid chromatography analysis. The developed method had acceptable recoveries (86.8-98.6%, n = 3) and precision (2.7-4.6%) at three spiked levels (0.05-0.5 µg g(-1)).

Thermoresponsive ketoprofen-imprinted monolith prepared in ionic liquid.

A thermoresponsive imprinted monolith with the ability of molecular recognition for ketoprofen was prepared for the first time. The smart monolith was synthesized in a stainless steel column using acrylamide (AAm) and 2-acrylamide-2-methyl propanesulfonic acid (AMPS) as functional monomers, which can form interpolymer complexation to restrict access of the analyte to the imprinted networks at low temperatures. To avoid a high back pressure of the column derived from neat dimethyl sulfoxide (DMSO) as a porogenic solvent that is needed to solve polar AMPS, an ionic liquid, [BMIM]BF4, was introduced into the pre-polymerization mixture. The molecular recognition ability towards ketoprofen of the resulting thermoresponsive molecularly imprinted polymer (MIP) monolith displayed significant dependence on temperature compared with a non-imprinted column (NIP), and the greatest imprinting factor was achieved at the transition temperature of 35 °C (above 10). Furthermore, the number of binding sites of the smart MIP monolith at 35 °C was about 76 times as large as that at 25 °C. In addition, Freundlich analyses indicated that the thermoresponsive MIP monolith had homogeneous affinity sites at both 25 and 35 °C with heterogeneity index 0.9251 and 0.9851, respectively.

Synthesis and theoretical study of molecularly imprinted monoliths for HPLC.

Molecularly imprinted monoliths integrate the high permeability of monolithic materials and the high selectivity and affinity of molecularly imprinted polymers (MIP). Thus, in recent years, development of this novel MIP format in HPLC has expanded quickly, particularly use of organic materials. This review focuses on the principal aspects of good practice in polymerization, theoretical studies, and recent developments in molecularly imprinted monoliths. Some thoughts on perspectives of MIP monoliths are also expressed.

Rapid preparation of molecularly imprinted polymer by frontal polymerization.

Frontal polymerization was successfully applied, for the first time, to obtain molecularly imprinted polymers (MIPs). The method provides a solvent-free polymerization mode, and the reaction can be completed in 30 min. By this approach, MIPs were synthesized using a mixture of levofloxacin (template), methacrylic acid, and divinylbenzene. The effect of template concentration and the amount of comonomer on the imprinting effect of the resulting MIPs was investigated. The textural and morphological parameters of the MIP particles were also characterized by mercury intrusion porosimetry, nitrogen adsorption isotherms, and scanning electron microscopy, providing evidence concerning median pore diameter, pore volumes, and pore size distributions. The levofloxacin-imprinted polymer formed in frontal polymerization mode showed high selectivity, with an imprinting factor of 5.78. The results suggest that frontal polymerization provides an alternative means to prepare MIPs that are difficult to synthesize and may open up new perspectives in the field of MIPs.

Preparation and characterization of an imprinted monolith by atom transfer radical polymerization assisted by crowding agents.

A method based on reverse atom transfer radical polymerization (R-ATRP) and molecular crowding has been used for design and synthesis of monolithic molecularly imprinted polymers (MIPs) capable of recognizing ibuprofen (IBU). 4-Vinylpyridine (4-VP) was used as the functional monomer, and ethylene glycol dimethacrylate (EDMA) was the crosslinking monomer. Azobisisobutyronitrile (AIBN)-CuCl(2)-N,N,N',N",N"-pentamethyldiethylenetriamine (PMDETA) was used as the initiating system. Compared with conventional radical polymerization-based IBU-MIPs, the imprinting effects of the obtained IBU-MIPs was enhanced, suggesting the merit of combination of reverse ATRP and molecular crowding. In addition, it was found that the polymerization time of the molecularly imprinted monolithic column, the amount of template, the degree of crosslinking, and the composition of mobile phase greatly affected retention of the template and the performance of molecular recognition.

Preparation of carbon nanotubes and polyhedral oligomeric-reinforced molecularly imprinted polymer composites for drug delivery of gallic acid.

In this paper, an enhanced imprinting effect of utilizing single wall carbon nanotubes (SWCNT) and polyhedral oligomeric silsesquioxanes (POSS) was suggested to improve drug delivery. The combination of 1-butyl-3-methylimidazoliumtetrafluoroborate ([BMIM]BF4), tetrahydrofuran (THF) and choline chloride-ethylene glycol (ChCl / EG) was used as a ternary porogen to prepare molecularly imprinted polymer (MIPs) doped with SWCNT and POSS. In the presence of gallic acid (GAL), 4-vinylpyridine (4-VP) and ethylene glycol dimethacrylate (EDMA) were used as functional monomer and crosslinker, respectively. The structure and morphological parameters of the MIP composite, such as surface area and pore size distribution, were also measured. In the studies of in vitro releases, superior controlled release characteristics can be achieved due to the enhanced imprinting effect of the MIPs doped with POSS and SWCNT. In vivo release studies showed that the POSS-SWCNT MIP had the maximum plasma concentration after 4 h. Compared with the control MIPs and NIP, the POSS-SWCNT MIP displayed a maximum AUC0-9 of 544.73 (ng h mL-1), while only 327.48, 212.91, 230.35 and 275.13 (ng h mL-1) for the POSS MIP, SWCNT MIP, MIP and POSS-SWCNT NIP, respectively.

Improving identification of molecularly imprinted monolith to benzoylation modified peptides by a deep eutectic solvents monomer-induced cooperation.

In this study, a strategy of improving imprinting performance was developed using an enhanced cooperation effect of functional monomers based on deep eutectic solvents (DESs) monomer for the specific enrichment of benzoylation modified peptides. Zinc acrylate and DESs monomers were used as binary functional monomers, and ethylene glycol dimethacrylate was used as the cross-linking agent with SGRGKbz as template to prepare an imprinted monolith. It was observed that the use of DESs monomer significantly improveed the affinity of benzoylation imprinted monolith and increased the adsorption capacity. Under optimal conditions, the recovery and imprinting factor (IF) of the imprinted monolith for SGRGKbz can reach 93.0% and 10.58, respectively. The average recovery of SGRGKbz extracted from the spiked histone digestion solution can reach 88.4% (n = 5, RSD = 3.4%). After treatment with the benzoylation imprinted monolith, 12 benzoylation modified peptides, 13 benzoylation modified sites and 12 benzoylation proteins could be identified in the digestion of mouse liver protein, while only one of each benzoylation modified peptide, benzoylation modified site and benzoylation protein could be identified in the untreated digestion of mouse liver protein. The results indicated that the prepared imprinted monolith using DESs-based functional monomer was an effective method to increase the affinity of the resulting MIP.

Origin of macromolecular crowding: Analysis of recognition mechanism of dual-template molecularly imprinted polymers by in silico prediction.

Multi-template imprinting is one of the challenge for molecular imprinting since the selectivity and binding affinity for each analyte decrease significantly compared with the corresponding molecularly imprinting polymers (MIPs) against single template. In this work, molecular crowding effect was tried to remedy the problem of imprinting reduction caused by the competition of two templates. Methacrylic acid (ACR) was used as functional monomer, ethylene dimethacrylate (EDMA) as crosslinker, and polystyrene (PS) as macromolecular crowding agent. With levofloxacin (S-OFX) as the first template, a number of compounds with varied chemical structure were chosen as the second template to investigate the imprinting effect of dual-template. When S-OFX and naproxen (S-NAP) was used as the dual-template, the imprinting factor (IF) of the resulting MIP for S-OFX was 20.1 and IF for S-NAP was 10.9. In contrast, for the single-template MIPs, IF for S-OFX was 22.4, and IF for S-NAP was 11.9. As a comparison, the IF of the DT-MIP prepared in absence of PS was only 2.3 for S-OFX and 1.0 for S-NAP. To analyze recognition mechanism of the molecular crowding-based imprinting system, molecular dynamics simulations to the chain structure of PS and binding modes between template and functional monomers was conducted by NAMD software. All the results displayed that molecular crowding is a promising method to improve the affinity of the dual-template imprinted polymer.

CEC separation of ofloxacin enantiomers using imprinted microparticles prepared in molecular crowding conditions.

Molecular crowding is a new concept to obtain molecularly imprinted polymers (MIPs) with greater capacity and selectivity, which could shift the equilibrium of a print molecule reacting with functional monomers in the direction of complex formation side. In this work, molecular crowding agent was first applied to the preparation of MIPs microparticles by precipitation polymerization. A new system of molecular crowding surrounding was developed, composed of polystyrene and tetrahydrofuran, in the presence of the template (S)-ofloxacin. Partial filling capillary electrochromatography (CEC) was utilized to evaluate imprinting effect of the resulting microparticles by chiral separations of ofloxacin. Some important parameters in the preparation, i.e. template to monomer ratio, influence of cross-linking monomers and functional monomer composition on the CEC separation of MIP microparticles were investigated. Baseline separation of ofloxacin (R(s) =1.53) was obtained under optimized conditions and the highest theory plate of the later eluent (S)-ofloxacin was 5400. The textural and morphological parameters for imprinted particles, such as Brunauer-Emmett-Teller surface areas, pore volumes and pore size distributions have also been determined. Compared to the MIP microparticle prepared by conventional precipitation polymerization, the (S)-ofloxacin-imprinted particles formed under molecular crowding conditions showed higher selectivity (α=1.09) and separation efficiency (<25 min) in the CEC mode.

Recent developments and applications of molecularly imprinted monolithic column for HPLC and CEC.

As a new generation of stationary phases, monolithic supports have attracted significant interest in high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) because of their ease of preparation, high reproducibility, versatile surface chemistry and rapid mass transport. Molecularly imprinted polymers (MIPs) are synthetic materials with high specific recognition ability to template molecule. The combination of monolithic column and MIPs integrates the high efficiency of modern chromatography and the high selectivity provided by MIPs. This review focuses on the recent developments and applications of all kinds of monolithic matrix, i.e. organic polymer-based and silica-based MIP monolith in HPLC and CEC mode.

[Preparation of liquid crystal-based molecularly imprinted monolith and its molecular recognition thermodynamics].

Molecularly imprinted polymers (MIPs) incorporated with liquid crystalline monomers can imprint and recognize templates at a very low level of crosslinking, thus addressing challenges associated with conventional MIPs, such as the embedding of the imprinted sites, low binding capacity, and slow mass transfer due to the high degree of crosslinking. Compared with traditional MIPs, the prepared MIPs have a greater number of easily binding sites, which can effectively overcome the embedding and low utilization of imprinting sites. Simultaneously, with a decrease in the level of chemical crosslinking, the mass transfer of template molecules can be significantly improved. However, the imprinting effect of liquid crystalline MIPs is generally weaker than that of traditional MIPs due to the low degree of crosslinking. Therefore, to obtain liquid crystalline MIPs with a good imprinting effect, a series of low-crosslinked liquid crystalline molecularly imprinted monoliths were prepared by graft polymerization and evaluated by high performance liquid chromatography (HPLC) to systematically determine the relation between the polymerization parameters and the affinity of the resulting liquid crystalline MIPs. In this experiment, trimethylolpropane trimethacrylate (TRIM) was used to synthesize a monolithic column skeleton with toluene and dodecyl alcohol as porogens. (S)-Naproxen was used as a template and liquid crystalline monomer 4-(4-cyanophenyl)-cyclohexyl ethylene (CPCE) was added for grafting to synthesize the liquid crystalline MIP monolith. The influence of the acetonitrile content and pH in the mobile phase on the chromatographic retention of the template molecule was investigated. The results showed that the main force of MIP recognizing naproxen changed from hydrogen bonding to hydrophobic interaction by the addition of the liquid crystalline monomer. Frontal analysis and adsorption isotherm fitting, including Langmuir, Freundlich, and Scatchard fitting, showed that when the crosslinking degree was 15%, the liquid crystalline MIPs exhibited the highest imprinting factor and heterogeneity, and the specific adsorption was stronger than non-specific adsorption. By analyzing the stoichiometric displacement model, the total affinity of the MIP monoliths for the template molecules (ln A) was determined to be 0.645, significantly higher than that of its analogues, indicating that the liquid crystalline imprinted monolith had a higher total affinity for the template molecule. The spatial matching degree (nß) of the template molecule to the cavity structures of MIPs was also very high, and only inferior to that of ketoprofen. Nevertheless, the ln A value of ketoprofen was only 0.242, which indicated that the spatial effect was not the key factor in determining the recognition ability of liquid crystalline imprinting systems. An analysis of the separation thermodynamics revealed that the separation of the liquid crystalline MIPs was an entropy-controlled process, while that of conventional liquid crystalline-free MIPs was an enthalpy-controlled process. Based on the above results, the addition of a liquid crystalline monomer may alter the recognition mechanism of MIPs, and an appropriately low crosslinking degree can significantly improve the recognition performance of liquid crystalline MIPs, paving the way for a new generation of MIPs.

Improving performance of molecularly imprinted polymers prepared with template of low purity utilizing the strategy of macromolecular crowding.

The topic in the present paper is to prepare molecularly imprinted polymer (MIP) using the template molecule with low purity. For the first time, a surrounding of macromolecular crowding was established to promote the formation of the complex of the template with functional monomer efficiently thus highly pure template molecule was unnecessary. In this study, the MIP monolith was synthesized using low purity lactucopicrin as template in place of highly pure one, and polystyrene was used as macromolecular crowding agent. 4-Vinylpyridine and ethyleneglycol dimethacrylate were used as functional monomer and crosslinker, respectively. Polymerization parameters, including the ratio of functional monomer/template, various template concentrations, and PS concentration on the affinity of the resulting MIP were systematically investigated. For the lactucopicrin MIP made with the purity of lactucopicrin of 92%, the imprinting factor can be up to 2.2. The resulting MIP was filled in solid phase extraction (SPE) cartridge to purify lactucopicrin from the crude extract of Cichorium glandulosum Boiss. et Huet. After two cycles of MIP SPE for the crude extract, the highest recovery and purity of lactucopicrin was 64.8% and 97.8%, respectively. The results indicated that the use of macromolecular crowding agent is an effective method for improving the performance of the MIP prepared with the template of low purity, particularly valuable to the cases in which the highly pure target molecule is hard to be obtained.