Preparation and Application of Molecularly Imprinted Polymers for Alkaloids: Review

Molecularly imprinted polymers (MIPs) prepared by molecular imprinting technology (MIT) are polymers with specific recognition sites matching the shape, size and functional groups of template molecules, which can selectively identify and enrich target analytes (template molecules), and have been widely used in sample pretreatment, chemical/biological sensing and other fields. However, in the processes of preparation and applications of MIPs, there are still some problems, such as difficult elution of template molecules, fewer effective recognition sites, low binding capacity, low mass transfer rate and poor recognition in aqueous media. As a multidisciplinary technology, MIT has developed rapidly by borrowing and integrating related advanced technologies/strategies of other fields. Consequently, a variety of new imprinting technologies and strategies have continuously emerged, which not only effectively solve the abovementioned problems but also push forwards the development of novel MIPs and widen their applications. In this paper, orienting the applications of MIPs in sample pretreatment, sensors and stimuli responses, some advanced preparation technologies and strategies for MIPs materials are highlighted, including ingenious imprinting technologies (surface imprinting, nanoimprinting; controlled/living polymerization, solid-phase synthesis, etc.), special imprinting strategies (multi-template/monomer imprinting, dummy imprinting, boronate affinity imprinting, etc.), and stimuli-responsive imprinting (magnetic, temperature, pH responsive, etc.). Fundamental features of the advanced imprinting technologies/strategies and their utilizations for MIPs preparations along with representative applications are described in details, involving important issues and research challenges. Firstly, a comprehensive overview of main imprinting technologies and strategies for MIPs preparation in sample pretreatment application is provided. In this regard, MIPs are used as selective adsorbents of various extraction technologies such as solid-phase extraction (SPE), dispersive SPE and magnetic SPE. Aiming at high selectivity and high adsorption capacity, the MIPs should have ideal morphology, uniform size and excellent surface properties. Besides conventional preparative methods, it is required to introduce new imprinting technologies and strategies, mainly including the ingenious imprinting technologies of surface imprinting, nanoimprinting, controlled/living free radical polymerization (CLRP), click chemistry, hollow porous polymer synthesis technology and solid-phase synthesis, and the special imprinting strategies of multi-template/monomer imprinting, dummy/segment imprinting, magnetic material and boronate affinity imprinting. Surface imprinting and nanoimprinting technologies are usually adopted by coupling with the abovementioned imprinting technologies and strategies. Secondly, advanced imprinting technologies and strategies for the construction of MIPs-based sensors are summarized. For the sensors, the MIPs as recognition elements can specifically bind target analytes and as transduction elements can generate output signals for detection. Typically, the output detection signals can be classified into three types, electrochemical, optical and piezoelectric types according to the transduction mechanism; molecular imprinting based electrochemistry, fluorescence and surface enhanced Raman scattering sensors are the research hotspots. For sensing applications, it is necessary to consider the main parameters such as response time, linear dynamic range, sensitivity, selectivity and reproducibility. Therefore, the MIPs should have excellent interface properties by employing appropriate preparative technologies and strategies. Nanoimprinting, surface imprinting and composite material imprinting strategy have become the preferences. Herein, constructions of molecular imprinting fluorescence sensors are emphatically introduced, especially ratiometric fluorescence ones. Thirdly, the imprinting technologies and strategies orienting stimuli-responsive application are briefly introduced, for preparing stimuli-responsive MIPs (SR-MIPs) with specific recognition ability toward targeted molecules under stimuli-regulation and thereby achieving intelligent control. SR-MIPs are able to sensitively respond to specific external physicochemical/biological stimuli with a considerable and reversible change in their properties, such as molecular chain structure, solubility, swelling or dissociation behavior, resulting in regular changes of imprinting properties. The most reported magnetic, temperature, photonic and pH sensitive SR-MIPs and their dual or multi stimuli responsive SR-MIPs are reviewed. The rapid development of smart ecofriendly SR-MIPs and their stimuli-responsive application will accelerate the drug delivery application and assist the widely carried out sample pretreatment and sensors applications. Lastly, future perspectives of MIT and MIPs are proposed. In order to solve the core issues of selectivity, mass transfer rate and adsorption capacity of MIPs, it is imperative to rationally combine various imprinting technologies and strategies. The ingenious fusion of MIT and various advanced technologies should be continuously strengthened to promote the preparation of MIPs materials.

The separation and detection of flavonoids from various natural products have attracted increasing attention in the field of natural product research and development. Depending on the high specificity of molecularly imprinted polymers (MIPs), MIPs are proposed as efficient adsorbents for the selective extraction and separation of flavonoids from complex samples. At present, a comprehensive review article to summarize the separation and purification of flavonoids using molecular imprinting, and the employment of MIP-based sensors for the detection of flavonoids is still lacking. Here, we reviewed the general preparation methods of MIPs towards flavonoids, including bulk polymerization, precipitation polymerization, surface imprinting and emulsion polymerization. Additionally, a variety of applications of MIPs towards flavonoids are summarized, such as the different forms of MIP-based solid phase extraction (SPE) for the separation of flavonoids, and the MIP-based sensors for the detection of flavonoids. Finally, we discussed the advantages and disadvantages of the current synthetic methods for preparing MIPs of flavonoids and prospected the approaches for detecting flavonoids in the future. The purpose of this review is to provide helpful suggestions for the novel preparation methods of MIPs for the extraction of flavonoids and emerging applications of MIPs for the detection of flavonoids from natural products and biological samples.

BackgroundCyanazine (CYZ) is one of the triazine herbicides to prevent broadleaf grass and weeds in crops. Despite its affordability and productivity in increasing crop yield, the extensive usage of CYZ contributes to environmental pollution and poses risks to living organisms. Most research has focused on detecting CYZ in the environment and its toxicity to humans and the ecosystem. For these reasons, molecular imprinting technology (MIT) can be applied to produce an effective adsorbent material of high binding affinity and selectivity towards its target compound which is known as molecularly imprinted polymers (MIPs). In this study, MIP was prepared by precipitation polymerization using CYZ as a template molecule, methacrylic acid (MAA), acrylamide (AAm) and 4-vinylpyridine (4VP) as functional monomers, and ethylene glycol dimethacrylate (EGDMA) as cross-linker in the ratio of 1:6:12, respectively. The effects of contact time, initial concentration, pH, and polymer dosages on the adsorption efficiencies of MIPs were also investigated in this study.ResultsMIPs of CYZ were successfully synthesized by precipitation polymerization method with a non-covalent approach using different functional monomers such as methacrylic acid (MAA), acrylamide (AAm) and 4-vinylpyridine (4VP). For the comparison study, the non-imprinted polymer (NIP) was synthesized without the addition of CYZ, the template molecule. The FTIR analysis indicated the interactions among CYZ and functional monomers (MAA, AAm or 4VP) in the presence of EGDMA as a cross-linker. The FESEM analysis showed that only MIP (AAm) and NIP (AAm) had regular and spherical polymer particles while MIP (MAA), NIP (MAA), MIP (4VP) and NIP (4VP) were agglomerated and irregular in shape. The EDX analysis showed that the MIPs were mainly composed of carbon and oxygen. Meanwhile, the BET analysis of MIP (AAm) had higher surface area, total pore volume and average pore radius than that NIP (AAm). Based on the batch binding study, MIP (AAm) (83.30%) had the highest binding efficiency than the MIP (MAA) (76.96%) and MIP (2VP) (76.90%) at a contact time of 240 min. The optimum conditions for the highest binding efficiency of MIP (AAm) were obtained at an initial concentration of 6 ppm, pH 7 and polymer dosage of 0.1 g polymer beads. The adsorption efficiency of MIP (AAm) with CYZ at the optimum parameters resulted in 86.39%. The selectivity test showed that MIP (AAm) was more selective towards CYZ than AME, the competitive compound with relative selectivity coefficient of 2.36. The kinetic isotherm of MIP (AAm) was best explained according to the pseudo-second-order kinetic model while the adsorption isotherm of MIP (AAm) was based on the Langmuir adsorption isotherm model. The MIP (AAm) was tested in the distilled water (DIW), tap water and river water spiked with CYZ and a substantial amount of CYZ was removed with a recovery of 86.67%, 84.75% and 84.69%, respectively.ConclusionThe MIPs of CYZ were successfully synthesized by the precipitation polymerization method using different functional monomers. Among those MIPs, MIP (AAm) showed the highest rebinding efficiency and therefore this MIP was selected for further studies. The best combination of CYZ, AAm was the main factor that contributed to the morphological and chemical properties, as well as the efficiency and selective binding performance of MIP (AAm). Since MIP (AAm) showed a substantial removal efficiency of CYZ in the environment specifically water sources, it has the capability to act as an adsorbent material for various purposes such as solid-phase extraction techniques and a stationary phase in various chromatographic techniques.Graphical

分子印迹技术（molecular imprinting technology， MIT）借鉴抗体-抗原特异性识别机制，能够高度精准地对目标物质进行选择性萃取，在分离、检测等领域极具应用潜力。但传统MIT在材料制备、样品前处理及检测分析中存在诸多亟待解决的问题：制备的分子印迹聚合物（molecularly imprinted polymers，MIPs）存在印迹位点不均一、模板分子残留严重、机械性能差等缺陷；以MIPs为吸附剂的前处理方法因目标物选择吸附速率慢而耗时，且特异性识别性能有待提升；基于MIPs的检测手段灵敏度低，检测耗时长，难以现场实时监测。这些问题制约了MIT的发展与广泛应用。近些年，电场辅助技术与MIT结合为解决上述问题提供了有效策略。制备MIPs时，在聚合体系中引入电场，使带电模板分子与功能单体受电场力定向移动，促使单体更有序地围绕模板分子排列，从而制得印迹位点均匀、分子取向性良好的MIPs。在样品前处理过程中，外部电场所提供的电泳驱动力可提升MIPs对目标物的传质速率，缩短吸附与解吸时间，优化其特异性识别性能。此外，MIPs电化学传感器的发展及其与微流控技术的结合显著提升了MIPs在检测领域的实用性。本文重点阐述电场在MIPs制备、样品前处理及检测分析三大关键环节的具体应用与作用机制，总结电场辅助MIT在环境监测、生物医学、食品安全等领域的应用前景，并展望了未来发展方向。

Molecularly imprinted polymers (MIP) exhibiting high selectivity and affinity tothe predetermined molecule (template) are now seeing a fast growing research. However,optimization of the imprinted products is difficult due to the fact that there are manyvariables to consider, some or all of which can potentially impact upon the chemical,morphological and molecular recognition properties of the imprinted materials. This reviewpresent a summary of the principal synthetic considerations pertaining to good practice in thepolymerization aspects of molecular imprinting, and is primarily aimed at researcher familiarwith molecular imprinting methods but with little or no prior experience in polymersynthesis. The synthesis, characteristic, effect of molecular recognition and differentpreparation methods of MIP in recent few years are discussed in this review, unsolvedproblems and possible developments of MIP were also been briefly discussed.

Molecularly imprinted polymers (MIPs) can specifically recognize template molecules in solution with imprinted cavities. Due to their capacity for scalable production, they can be used to isolate target products from natural products for industrial production in the fields of pharmaceuticals and food. In this study, magnetic single-template molecularly imprinted polymers (St-MIPs) instead of magnetic multi-template molecularly imprinted polymers (Mt-MIPs) were prepared by surface imprinting using Schizandrol A as a template molecule and deep eutectic solvent (DES) as a functional monomer, combined with solid-phase extraction (SPE) for the adsorption and separation of Schizandrol A, Schisantherin A, Schizandrin A, and Schizandrin B from Schisandra chinensis (Turcz.) Baill. (S. chinensis) fruits extracts. The synthesized MIPs were characterized by FT-IR, TEM, SEM, TG, XRD and VSM, and their adsorption properties were also evaluated. MIPs can specifically recognize the template molecules with high reusability. The purity of the total S. chinensis lignans after SPE was 74.05%, among which that of Schizandrol A, Schisantherin A, Schizandrin A, and Schizandrin B was 33.38%, 8.69%, 16.33% and 15.67%, respectively. Moreover, the one-step synthesis of carrier was easy to operate. And St-MIPs reduced the production cost compared with Mt-MIPs. This study provides a new idea for natural product separation by molecular imprinting technology (MIT).

Given continuous improvements in industrial production and living standards, the analysis and detection of complex biological sample systems has become increasingly important. Common complex biological samples include blood, serum, saliva, and urine. At present, the main methods used to separate and recognize target analytes in complex biological systems are electrophoresis, spectroscopy, and chromatography. However, because biological samples consist of complex components, they suffer from the matrix effect, which seriously affects the accuracy, sensitivity, and reliability of the selected separation analysis technique. In addition to the matrix effect, the detection of trace components is challenging because the content of the analyte in the sample is usually very low. Moreover, reasonable strategies for sample enrichment and signal amplification for easy analysis are lacking. In response to the various issues described above, researchers have focused their attention on immuno-affinity technology with the aim of achieving efficient sample separation based on the specific recognition effect between antigens and antibodies. Following a long period of development, this technology is now widely used in fields such as disease diagnosis, bioimaging, food testing, and recombinant protein purification. Common immuno-affinity technologies include solid-phase extraction (SPE) magnetic beads, affinity chromatography columns, and enzyme linked immunosorbent assay (ELISA) kits. Immuno-affinity techniques can successfully reduce or eliminate the matrix effect; however, their applications are limited by a number of disadvantages, such as high costs, tedious fabrication procedures, harsh operating conditions, and ligand leakage. Thus, developing an effective and reliable method that can address the matrix effect remains a challenging endeavor. Similar to the interactions between antigens and antibodies as well as enzymes and substrates, biomimetic molecularly imprinted polymers (MIPs) exhibit high specificity and affinity. Furthermore, compared with many other biomacromolecules such as antigens and aptamers, MIPs demonstrate higher stability, lower cost, and easier fabrication strategies, all of which are advantageous to their application. Therefore, molecular imprinting technology (MIT) is frequently used in SPE, chromatographic separation, and many other fields. With the development of MIT, researchers have engineered different types of imprinting strategies that can specifically extract the target analyte in complex biological samples while simultaneously avoiding the matrix effect. Some traditional separation technologies based on MIP technology have also been studied in depth; the most common of these technologies include stationary phases used for chromatography and adsorbents for SPE. Analytical methods that combine MIT with highly sensitive detection technologies have received wide interest in fields such as disease diagnosis and bioimaging. In this review, we highlight the new MIP strategies developed in recent years, and describe the applications of MIT-based separation analysis methods in fields including chromatographic separation, SPE, diagnosis, bioimaging, and proteomics. The drawbacks of these techniques as well as their future development prospects are also discussed.

Due to the specific recognition ability of the target molecules, molecularly imprinted polymers (MIPs) have been widely used in many fields. So far, the research in molecular imprinting mainly focused on two aspects: (1) synthesis of MIPs with more functions; (2) expanding applications of the traditional MIPs, especially for MIPs synthesized by precipitation polymerization. To address these points, our group recently introduced a new insight in molecular imprinting based on Pickering emulsion: (1) Pickering emulsion polymerization could be used to the synthesize water-compatible MIP spheres with well-controlled structures; (2) MIP nano/microparticles stabilized emulsions were introduced to molecular imprinting to endow the MIP particles with more functions, or to create new interfacial binding or catalytic systems for MIP nano/microparticles. It is noted that, although the first molecular imprinting in Pickering emulsion has been presented for only a few years, the publications in this field increased very fast. In this review, we summarized the synthesis of MIP spheres via Pickering emulsion polymerization, and discussed the advantages and limitations of this synthesis method. Due to the unique properties of the particle stabilized emulsion, the imprinted systems offered several advantages to the resulted MIP spheres, and which could be further utilized to solve the long-tern challenges in imprinting of small molecules: (1) well-designed hierarchical structures (surface nano-pores from the particle stabilizers and accessible molecular binding cavities from the templates); (2) group recognition of a series of chemicals with a similar moiety (epitope imprinting); and (3) specific recognition capability in aqueous conditions because of the hydrophilic surface of the MIPs. Moreover, for imprinting biomacromolecules and other large templates (virus particles, bacteria and animal cells), Pickering emulsion was also particularly interesting, because the templates themselves could be directly used as solid stabilizers to synthesis Pickering emulsion, and thus created accessible surface imprinted sites for the recognition. In this review, we also demonstrated that MIP nano/microparticles stabilized emulsions were promising interfacial systems in molecular imprinting. On one hand, this emulsion could be utilized for the fabrication of functional materials with pre-designed recognition ability towards the target molecules. As examples, the Janus MIP particles, which displayed attractive abilities as self-propelled transporters for controlled drug delivery, could be prepared via a wax-water Pickering emulsion. Multifunctional colloidosomes, which showed both specific molecular recognition of the MIP nanoparticles and dose-dependent fluorescence response to fructose, could be synthesized by using W/O Pickering emulsions stabilized by clickable nanoparticles: Alkyne-coated MIP nanoparticles and azide-modified nanoparticles, followed by an interfacial click reaction. On the other hand, the introduction of Pickering emulsion expanded tha application environment of the traditional MIP nano/microparticles to a oil-water interface. For example, MIP microgel stabilized Pickering emulsions with the capability to catalyze the formation of disulfide bonds in peptides at the O/W interface has been reported recently. These works system will greatly promote the application of molecular imprinting in various fields such as organic synthesis, disease diagnosis, proteomics and bio-imaging Moreover, we discussed the prospects of the applications of molecular imprinting in Pickering emulsion in a near future, (1) uniform MIP spheres should be fabricated by optimizing the emulsion systems and choosing suitable reactors; (2) stable Pickering emulsion systems should be developed for biomacromolecular imprinting, such as proteins, micro organisms and human cells; (3) MIP nano/microparticles prepared by Pickering emulsion should be designed for immunoassay, biomedicine, environmental governance and chemical catalysis. As a summary, we believed that molecular imprinting in Pickering emulsion provided a facile synthetic approach for producing new functional materials, as well as an interfacial binding or catalytic environment for appilication of the traditional MIP nano/microparticles. All these works show great potential in clinical pathogenic screening, noninvasive detection and drug delivery.

Molecular imprinting technology was employed to produce one kind of rhodamine B(RhB) molecularly imprinted polymer(MIP) microspheres by precipitation polymerization using RhB, acrylamide, ethylene glycol dimethacrylatea(EGDMA), azobisisobutyronitrile(AIBN), and acetonitrile as template, functional monomer, cross-linking agent, initiator and porogen, respectively. The resultant microspheres were characterized by scanning electron microscopy. The binding constant and chemical combination ratio of the complex formed between the template and the functional monomer were obtained by UV spectrophotometry, which showed that the RhB-monomer forms 1∶1 complexes and the binding constant is 5.3×103 L/mol. The effects of types and volumes of the porogen on the size and the uniformity of microspheres were also discussed. After packing the microspheres into column, it was used to absorb RhB from red pepper. The extraction conditions of molecular imprinting solid phase extraction(MISPE) column for RhB were optimized. Pepper samples spiked with RhB were extracted by MISPE column and analyzed by high performance liquid chromatography. The recoveries of MISPE column for RhB extraction were found to be 91.7%～103.5%, indicating the feasibility of the prepared MIPs for RhB extraction.

The abuse and residues of antibiotics have a great impact on the environment and organisms, and their determination has become very important. Due to their low contents, varieties and complex matrices, effective recognition, separation and enrichment are usually required prior to determination. Molecularly imprinted polymers (MIPs), a kind of highly selective polymer prepared via molecular imprinting technology (MIT), are used widely in the analytical detection of antibiotics, as adsorbents of solid-phase extraction (SPE) and as recognition elements of sensors. Herein, recent advances in MIPs for antibiotic residue analysis are reviewed. Firstly, several new preparation techniques of MIPs for detecting antibiotics are briefly introduced, including surface imprinting, nanoimprinting, living/controlled radical polymerization, and multi-template imprinting, multi-functional monomer imprinting and dummy template imprinting. Secondly, several SPE modes based on MIPs are summarized, namely packed SPE, magnetic SPE, dispersive SPE, matrix solid-phase dispersive extraction, solid-phase microextraction, stir-bar sorptive extraction and pipette-tip SPE. Thirdly, the basic principles of MIP-based sensors and three sensing modes, including electrochemical sensing, optical sensing and mass sensing, are also outlined. Fourthly, the research progress on molecularly imprinted SPEs (MISPEs) and MIP-based electrochemical/optical/mass sensors for the detection of various antibiotic residues in environmental and food samples since 2018 are comprehensively reviewed, including sulfonamides, quinolones, β-lactams and so on. Finally, the preparation and application prospects of MIPs for detecting antibiotics are outlined.

Molecular imprinting is an emerging technology that allows to introduce nanostructured cavities into a polymer. In preparing molecular imprinted polymers (MIPs), the functional monomer(s) is first prearranged around the template molecule by specific interactions; the polymerisation is then carried out with a high percentage of cross-linking agent (which “freezes” the macromolecular network). Molecular mechanics and dynamics can be used to gain indications on the best monomers to be used in order to maximize interactions with the template. Once the polymerization reaction has been completed, the template is removed from the rigid three-dimensional network, leaving free recognition cavities available for the successive selective rebinding of the template itself. Precipitation polymerisation in dilute solutions involves the spontaneous formation of submicron scale polymer particles, which result suitable for recognition-rebinding application. Therapeutic applications: The recognition mechanism by MIPs relies mainly on the establishment of reversible hydrogen bonding interactions. It is clear that the efficiency of this mechanism is endangered in aqueous environments. MIPs working in water solutions are clearly of great interest in the medical and food industry and in sensor applications. We recently overcame these difficulties by the realisation of a system where cross-linked MI methylmethacrylate-methacrylic acid nanospheres where loaded on the surface or inside the matrix of porous membranes created by phase inversion. E.g. membranes were modified by adding cholesterol imprinted nanoparticles. Rebinding performances of nanoparticles modified membranes in buffer solution were tested showing a specific recognition of 14.09 mg of cholesterol/g of system (membrane and nanoparticles), indicating maintained binding capacity of supported particles as well. Tissue engineering: The engineering of functionalised polymeric structures for the study of cell activity is essential to the development of biological substitutes containing vital cells capable of regenerating or enhancing tissue function. Cells are organised within a complex matrix consisting of high molecular weight protein and polysaccharides known as the Extracellular Matrix (ECM). Two approaches are described to explore the possibility to provide scaffolds with specific and selective recognition of peptide sequences or proteins involved in cell adhesion mechanisms: one approach consists in the modification of porous structures with nanoparticles imprinted with aminoacid sequences (epitopes) of ECM proteins or transmembrane integrins, while the other consists in the combination of Soft Litography and Molecular Imprinting technologies (SOFT-MI). This technology allows to create imprinting nanocavities selective towards ECM proteins in microfabricated scaffolds, and in particular it permits to realise patterns with a well defined microscale geometry in polymethylmethacrylate (PMMA) scaffolds providing them with cell adhesion properties that were missing in the non-imprinted scaffold.

Molecular imprinting technology (MIT) is a developing technique with high recognition which is just like the recognition between enzymes and antibodies in the organism. Molecularly imprinted polymers (MIPs), synthetic materials obtained using the imprinting technology, have played a huge advantage and been used in many fields. Especially, MIPs have been applied to the extraction and separation of analytes as the selective adsorbent of solid-phase extraction (SPE), which is known as molecularly imprinted polymer solid-phase extraction (MISPE) in recent years. In the present review, the methodology of MIPs preparation and evaluation are explained. Moreover, recent great developments of SPE and MISPE are discussed, and the potential application of MISPE in extraction of traditional Chinese medicine (TCM) active ingredients are also presented briefly.

Molecularly imprinted polymers (MIPs) are synthetic materials able to selectively rebind a target molecule in preference to other closely related compounds. Molecular imprinting technology involves the synthesis of MIPs in the presence of a template with the aim of introducing complementary and selective binding sites into the polymeric matrix. This synthetic approach allows us to obtain stable polymeric materials characterized by significant molecular recognition abilities, as well as low cost and easy preparation, and, at the same time, resistance to a wide range of conditions such as pH, organic solvents, temperature and pressure. Currently, MIPs are attracting considerable research interest due to their potential applications in several fields including separation sciences, solid-phase extraction (SPE), biotechnology, catalysis, chemo/biosensors and drug delivery. Stimuli-responsive polymers can be defined as intelligent materials able to respond to specific environmental stimuli, such as the presence of another molecule, pH, temperature, magnetic field and irradiation, with consequent changes in their properties. Stimuli-responsive MIPs combine the selective recognition properties for a template molecule with the ability to respond to external stimuli. Therefore, this chapter aims at an overview of molecular imprinting technology, the design of MIPs and the synthetic approaches with particular attention devoted to the field of stimuli-responsive MIPs.

Synthetic approaches to parabens molecularly imprinted polymers and their applications to the solid-phase extraction of river water samples

Separation and screening of compounds of biological origin using molecularly imprinted polymers