Application of molecularly imprinted polymers-based sensors for determination of acute coronary syndrome biomarkers

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.

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。

极性农药包括杀菌剂、除草剂、杀虫剂等,种类丰富,成本低廉,在农业中应用广泛,其滥用易导致水资源和土壤等环境污染,人类通过间接接触动植物源性食品和环境中的极性农药残留也增加了农药暴露风险。极性农药的物理化学性质差异大,通常痕量存在于食品和环境样品等复杂基质中,这对其准确检测分析带来了挑战。分子印迹聚合物(MIPs)作为一种人工制备的选择性吸附剂,具有与模板分子在空间结构、大小尺寸和功能基团上互补的特定识别位点,且易于制备,成本低,稳定性好,重复利用率高,已被广泛用于极性农药残留的样品前处理和分析检测中。MIPs可以作为固相萃取(SPE)、固相微萃取(SPME)、磁性固相萃取(MSPE)、搅拌棒固相萃取(SBSE)等前处理方法的吸附剂,还可用于制备光、电、化学传感器,作为质谱检测的离子源基底和拉曼光谱的增强基底。目前针对极性农药残留的检测,已有许多研究报道了多种分子印迹材料用于高效分离分析各种复杂基质中的极性农药残留,但未见此方面的综述报道。该文首先介绍了MIPs的印迹策略、聚合策略,并针对传统MIPs制备和应用中存在的问题,简要概括了一些新型的分子印迹策略和制备技术;然后从极性农药残留分析的角度出发,总结归纳了分子印迹材料近年来特别是近5年来在各种极性农药残留(包括新烟碱类、有机磷类、三嗪类、唑类、脲类等)检测中的应用,并针对现存问题展望了其未来的发展方向和趋势。

Molecular imprinting technology is a new analytical method that is highly selective and specific for certain analytes in artificial receptor design. The renewal possibilities of this technology make it an ideal material for sundry application fields. Molecularly imprinted polymers (MIPs) are polymeric matrices that have molecules printed on their surfaces; these surfaces can chemically interact with molecules or follow the pattern of the available template cavities obtained using imprinting technology. A MIP is useful for separating and analysing complex samples, such as biological fluids and environmental samples, because it is a strong analytical recognition element that can mimick natural recognition entities like biological receptors and antibodies. The MIP components consist of the target template, functional monomer, crosslinker, polymerisation initiator, and porogen. The effectiveness and selectivity of a MIP are greatly influenced by variations in the components. This review will provide an overview of the effect of MIP component ratio on analytical performance to each target analyte; it will also provide a strategy to obtain the best MIP performance. For every MIP, each template : monomer : crosslinker ratio shows a distinct performance for a specific analyte. The effects of the template : monomer : crosslinker ratio on a MIP's analytical performances-measured by the imprinting factor, sorbent binding capacity, and sorbent selectivity-are briefly outlined.

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

Specific recognition of tyrosine-phosphorylated peptides by epitope imprinting of phenylphosphonic acid

液晶分子印迹聚合物(MIPs)因刚性液晶单体的加入而在超低交联度水平下也能印迹和识别模板分子,有效解决了传统MIPs因高交联度造成的位点包埋、结合容量低、传质慢等问题。尽管液晶MIPs具有如此独特的优势,但却面临着由于交联度的大幅度降低而导致印迹效果下降的问题。为了研究液晶MIPs的结合特性,制备具有良好印迹效果的低交联液晶MIPs,该文通过二次接枝聚合,制备了一系列不同交联度的液晶分子印迹整体柱,用高效液相色谱法研究了聚合参数与印迹整体柱亲和性的关系。实验中选用三羟甲基丙烷三甲基丙烯酸酯(TRIM)为交联剂,以甲苯和十二醇为致孔剂合成整体柱骨架,并在此基础上以(S)-萘普生为模板,加入液晶单体4-氰基苯基单环己基乙烯(CPCE)进行二次聚合接枝。实验中系统考察了流动相中乙腈比例及缓冲液pH值对色谱保留的影响,结果发现液晶单体的加入使得MIPs对萘普生保留控制机制由原来的氢键作用变为了疏水作用;通过动态吸附实验得到的突破曲线经前沿分析及对吸附等温线Langmuir、Freundlich和Scatchard分析拟合,发现交联度为15%时液晶MIPs印迹因子最大(3.78)、非均一性最强,且特异性吸附量高于非特异性吸附量。液晶MIPs的计量置换模型(SDM-R)分析表明,液晶印迹整体柱对模板分子的总亲和力(ln A=0.645)明显高于其类似物;而从空间匹配程度看,与液晶印迹整体柱空间匹配程度最高的是酮洛芬而非模板分子,但液晶印迹整体柱对酮洛芬的总亲和力(ln A=0.242)不及模板分子的一半,表明在本低交联液晶印迹系统中,空间效应不是决定印迹系统识别能力的主要因素。进一步的分离热力学研究发现,低交联液晶印迹柱的|ΔΔH|<T|ΔΔS|,而交联度为70%的非液晶MIPs柱的|ΔΔH|>T|ΔΔS|,表明液晶MIPs的分离过程是一个熵控制过程,而常规无液晶MIPs的分离过程是一个焓控制过程。上述结果表明,液晶单体的加入改变了MIPs的识别机制,适当的低交联度可显著提高液晶MIPs的识别性能,因此液晶MIPs这些特质有望使其成为新一代的MIPs。

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.

Chapter 3 - Parameters that affect molecular imprinting polymers

Chapter 3 - Inefficient removal of templates as a limitation for molecular imprinting of polymers

A Biosensor Combining Molecularly Imprinted Polymers (M-MIPs) and Surface Enhanced Raman Spectroscopy (SERS) to Detect Antibiotics in Food Samples &nbsp; Yi Sun,1*, Jon Ashley1, Kaiyu Wu1, and Anja Bosen1. 1 Department of Micro- and Nanotechnology, Technical University of Denmark, &Oslash;rsteds Plads, DK-2800 Kgs. Lyngby, Denmark &nbsp; * Email: sun.yi@nanotech.dtu.dk; Tel.: +45 45256319 &nbsp; In this study, temperature-responsive magnetic molecularly imprinted polymers (M-MIP) nanoparticles were synthesized for the first time for the extraction of cloxacillin in pork products. By combining the M-MIPs with surface enhanced Raman spectroscopy (SERS), a sensitive biosensor was demonstrated to detect cloxacillian with pico-mole sensitivity. MIPs are synthetic ligands which can be tailored to bind any analyte of choice1.&nbsp; They are of great interest due to their thermal stability, robustness, low cost and comparable binding affinity. They have been used in sample preparation and biosensing as an attractive alternative to natural antibodies to capture targets ranging from small molecules to big proteins. &nbsp; In this work, the magnetic nanoparticles with MIP-based receptors were synthesized for efficient and rapid extraction of antibiotic residues in pork samples. &nbsp;Fe3O4 nanoparticles were obtained&nbsp;using the solvothermal synthesis.&nbsp; The resultant nanoparticles were treated with Tetraethyl orthosilicate (TEOS) to form a SiO2&nbsp;layer.&nbsp; Finally a thin MIP layer was polymerized round the nanoparticles using azobisisobutyronitrile (AIBN) as the initiator, ethylene glycol dimethacrylate (EDGMA) as the cross-linker, N-isopropylmethacryamide (NIPAm), methacrylic acid (MAA) as the monomers and the antibiotic as the template.&nbsp; By adding the monomer NIPAm, the MIPs become temperature responsive, and can swell at low temperature to release the target. The corresponding magnetic non-imprinted polymer nanoparticles (M-NIP) was prepared using the same method in the absence of the template. An Overview of the synthesis strategy is shown in Fig. 1. The resultant M-MIP nanoparticles were characterized using IR, XRD scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Fig. 2).&nbsp; Both binding affinities of the resultant M-MIPs and M-NIPs were tested using UV absorbance (Fig. 3). M-MIPs with 300-400 nm in size and good binding capacities were obtained. To demonstrate the feasibility of using M-MIPs for sample preparation, the synthesized M-MIPs were mixed with pork blood samples spiked with Chloxacillian. After incubation at room temperature, the M-MIPs were collected using a magnet and washed by acetonitrile. Owing to the thermos-responsive properties of MIPs, Chloxacillian was easily released by cooling the MIPs to 4 degree. The collected Chloxacillian was dropped on a SERS substrate which contained an array of silicon micropillars coated with silver. The corresponding calibration plots showed a detection limit (LOD) of about 50 pmol (Fig. 4). The biosensor combining M-MIPs and SERS would be widely used on site or in the field for rapidly screening food contaminants to ensure food safety. Fig. 1: Overview of the synthesis of M-MIPs &nbsp; &nbsp; Fig.2 IR characterization of Fe3O4, Fe3O4@SiO2, Fe3O4@ SiO2-MPA and, Fe3O4@SiO2-MIP; XRD of Fe3O4. Fig.3 (A) Binding kinetics and (B) Binding capacity of Cloxacillian MIPs and NIPs. &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Fig.4 SERS spectra of cloxacillin in MeOH:acetic acid (9:1) and corresponding calibration plots. &nbsp; REFERENCES: &nbsp; J. Ashley, M-A. Shahbazi, K. Kant, V. A. Chidambara, A.Wolff, D. D. Bang, Y. Sun, &ldquo;Molecularly Imprinted Polymers for Sample Preparation and Biosensing in Food analysis: Progress and Perspectives, Biosens. Bioelectron. 2017, 91, 606-615. &nbsp; &nbsp;

The versatility of molecularly imprinted polymers (MIPs) has led to their integration into applications like biosensing, separation, environmental monitoring, and drug delivery technologies. This diversity of applications has resulted in a plethora of synthesis approaches to precisely tailor the materials' properties to the specific demands. A critical, yet often overlooked, factor in MIP synthesis is the choice of porogen. Porogens play a pivotal role in defining the morphology, surface properties, swelling behavior, and binding efficiencies of the resulting MIPs. While aprotic solvents have traditionally been the standard in molecular imprinting, recent developments have expanded the variety of employed porogens accompanied by notable improvements in MIP performance. Therefore, this review aims to highlight both traditional and emerging types of porogens used in molecular imprinting, their influence on polymer properties and sorption performance, and their application across various sensing and extraction applications.

Biological receptors including enzymes, antibodies and active proteins have been widely used as the detection platform in a variety of chemo/biosensors and bioassays. However, the use of artificial host materials in chemical/biological detections has become increasingly attractive, because the synthetic recognition systems such as molecularly imprinted polymers (MIPs) usually have lower costs, higher physical/chemical stability, easier preparation and better engineering possibility than biological receptors. Molecular imprinting is one of the most efficient strategies to offer a synthetic route to artificial recognition systems by a template polymerization technique, and has attracted considerable efforts due to its importance in separation, chemo/biosensors, catalysis and biomedicine. Despite the fact that MIPs have molecular recognition ability similar to that of biological receptors, traditional bulky MIP materials usually exhibit a low binding capacity and slow binding kinetics to the target species. Moreover, the MIP materials lack the signal-output response to analyte binding events when used as recognition elements in chemo/biosensors or bioassays. Recently, various explorations have demonstrated that molecular imprinting nanotechniques may provide a potential solution to these difficulties. Many successful examples of the development of MIP-based sensors have also been reported during the past several decades. This review will begin with a brief introduction to the principle of molecular imprinting nanotechnology, and then mainly summarize various synthesis methodologies and recognition properties of MIP nanomaterials and their applications in MIP-based chemosensors. Finally, the future perspectives and efforts in MIP nanomaterials and MIP-based sensors are given.

Chapter 11 - Protein imprinting via epitope approach: An overview

A critical review of molecularly imprinted polymers for the analysis of organic pollutants in environmental water samples