Application of molecularly imprinted polymers as artificial receptors for imaging

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Application of molecularly imprinted polymers as artificial receptors for imaging Tereza Vaneckova , Jaroslava Bezdekova , Gang Han , Vojtech Adam , Marketa Vaculovicova PII: DOI: Reference:

S1742-7061(19)30747-0 https://doi.org/10.1016/j.actbio.2019.11.007 ACTBIO 6438

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Acta Biomaterialia

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16 July 2019 21 October 2019 4 November 2019

Please cite this article as: Tereza Vaneckova , Jaroslava Bezdekova , Gang Han , Vojtech Adam , Marketa Vaculovicova , Application of molecularly imprinted polymers as artificial receptors for imaging, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.11.007

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Application of molecularly imprinted polymers as artificial receptors for imaging

Tereza Vaneckova 1,2 , Jaroslava Bezdekova 1,2 , Gang Han 3 , Vojtech Adam 1,2 , Marketa Vaculovicova 1,2*

1

Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-

613 00 Brno, Czech Republic 2

Central European Institute of Technology, Brno University of Technology, Purkynova 123,

CZ-612 00 Brno, Czech Republic 3

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts

Medical School, Worcester, MA 01605, USA

* Corresponding author: Marketa Vaculovicova, Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mail: [email protected]; phone: +420-5-4513-3290; fax: +420-5-4521-2044

Synthetic materials with selective recognition properties toward templates are an alternative to traditionally used recognition biomolecules (e.g., antibodies). Key applications of molecularly imprinted polymers in imaging are highlighted and discussed with regard to the selection of the core material for imaging as well as commonly used imaging targets.

Table of Contents

Abstract ................................................................................................................................................... 4 Keywords ................................................................................................................................................ 4 1. Introduction ......................................................................................................................................... 6 2. Methods of MIP fabrication ................................................................................................................ 8 2.1. Preparation of polymers ............................................................................................................... 9 2.2. Initiation methods ....................................................................................................................... 10 2.3. Template imprinting methods..................................................................................................... 11 3.1. Luminescence imaging ............................................................................................................... 14 3.2. Alternative materials for imaging ............................................................................................... 17 4. Applications....................................................................................................................................... 18 4.1. Molecular targets ........................................................................................................................ 19 4.2. MIP-based imaging and therapy................................................................................................. 22 4.3. Pseudoimmunoassays ................................................................................................................. 26 5. Challenges of MIPs to be addressed .................................................................................................. 27 6. Conclusion and future perspectives ................................................................................................... 29 Acknowledgment............................................................................................................................... 30 Figure Captions ................................................................................................................................. 30 References ......................................................................................................................................... 43

Abstract Medical diagnostics aims at specific localization of molecular targets as well as detection of abnormalities associated with numerous diseases. Molecularly imprinted polymers (MIPs) represent an approach of creating a synthetic material exhibiting selective recognition properties toward the desired template. The fabricated target-specific MIPs are usually well reproducible, economically efficient, and stable under critical conditions as compared to routinely used biorecognition elements such as fluorescent proteins, antibodies, enzymes, or aptamers and can even be created to those targets for which no antibodies are available. In this review, we summarize the methods of polymer fabrication. Further, we provide key for selection of the core material with imaging function depending on the imaging modality used. Finally, MIP-based imaging applications are highlighted and presented in a comprehensive form from different aspects.

Keywords Luminescence; polymerization; microscopy; affinity

Medical diagnostics aims at specific localization of molecular targets as well as detection of abnormalities associated with numerous diseases. Molecularly imprinted polymers (MIPs) represent an approach of creating a synthetic material exhibiting selective recognition properties toward the desired template. The fabricated targetspecific MIPs are usually well reproducible, economically efficient, and stable under critical conditions as compared to routinely used biorecognition elements, e.g., antibodies, fluorescent proteins, enzymes, or aptamers, and can even be created to those targets for which no antibodies are available. In this review, we summarize the methods of polymer fabrication. Key applications of MIPs in imaging are highlighted and discussed with regard to the selection of the core material for imaging as well as commonly used imaging targets.

1. Introduction Specific localization and quantification of certain molecular targets is key for medical diagnostics and biology. In this context, bioimaging covers the determination of target molecules not only in and on cells and tissues but also in the whole body. To detect abnormalities or derive information about the function of the targets, determining their exact localization and following their dynamics are of particular interest. In routine analysis, fluorescent proteins or antibodies are commonly used. However, no versatile approach exists for the recognition and imaging of sites where recognition elements are very limited or not available. With this in mind, the application of tailor-made molecularly imprinted polymers (MIPs) shows to be a promising strategy. Molecular imprinting represents template-directed synthesis of polymers. The template is removed after complete polymerization, which reveals cavities that enable selective and sensitive capture and, in some cases, response to biological and chemical analytes through noncovalent bonds such as hydrogen bonding, hydrophobic interactions, ionic interactions, or van der Waals forces [1-3]. MIPs exhibit versatile recognition properties that are often comparable with those of natural molecules such as antibodies, enzymes, or aptamers, commonly exploited as biorecognition units [4]. Moreover, MIPs have the advantage of high stability and long-term reusability as well as cost-effectiveness [5]. However, the repeatability and accuracy of MIPs strongly depend on the fabrication strategies. In the field of imaging, nano-sized structures with a molecularly imprinted shell are a method of choice. Compared to the traditional planar structure of MIP, nanoparticle (NP)-based imprinted polymers possess several advantages: (1) Combination of a polymer matrix with nanostructures typically leads to increased sensitivity [6-8]. (2) Polymeric MIP-coated NPs can be synthesized to obtain a higher surface-to-volume ratio than thin films and thus substantially larger total surface area per unit weight of the polymer; geometric features

provide better accessibility of recognition sites for the analyte and lower mass transfer resistance. In addition to improved binding kinetics, the template can be more easily removed from the NPs, resulting in reduced template leaching [9-11]. In addition to these advantages of nano-sized MIPs, (3) a combination of the imprinted polymer and NP can be done by introducing a polymeric shell on the surface of the nanomaterial core with additional functionalities such as magnetic, semiconductive, or luminescent properties [12-14]. Similarly, diverse methods of detection have been associated with MIP-based systems with the most widespread mass-sensitive quartz crystal microbalance, electrochemical methods (potentiometric, impedometric, and cyclic voltammetric), and optical methods including luminescence, surface plasmon resonance (SPR), and surface -enhanced Raman scattering. According to Ma et al., luminescent MIP nanocomposites used for constructing platforms for sensing and separation can be further applied as signal transducers with molecular recognition properties, and they can potentially enhance the selectivity and sensitivity of the analytical methods [15]. This review summarizes the methods of fabrication of MIPs for imaging from the selection of the core material to polymerization methods. The overview is followed by selected applications of MIP-based imaging, both standard and hybrid ones. Potential improvements in MIPs in bioimaging applications are discussed in the last section.

2. Methods of MIP fabrication Molecular imprinting of synthetic polymers is a process of creation of the templates at the molecular level. Compared to biological antibodies, the production of MIPs is generally well reproducible, quick, and economical and does not involve animals. Likewise, the physical and chemical stability of the polymer enables the use of various solvents without damaging the recognition sites. Therefore, MIPs are potentially superior materials for cell imaging. Monomers play an important role in carrying functional groups with the ability to selfassemble around a template molecule (the target or a derivative). Next, copolymerization with cross-linking monomers results in the formation of a polymeric structure around the template. After template removal, the 3D binding sites in the polymer complementary to the template in size, shape, and functional groups are revealed. These binding sites exhibit affinity and selectivity often comparable to those of antibodies [4, 16]. In fact, MIPs can be used as a recognition element for any analyte, even to those targets for which the natural receptor or antibody does not exist. During recent years, methods of the formation of MIP NPs have been growing. The preparation of MIPs represents a very wide and variable topic. With focus on the fabrication strategies of polymers to be used in bioimaging, synthetic methods and methods of template imprinting are presented in this chapter. For better imagination and understanding, we divide the preparation of MIPs into three groups according to their method of preparation, initiation of polymerization, and template-imprinting strategy as shown in Fig. 1.

2.1. Preparation of polymers Favorable approaches for polymer preparation resulting in micro- or NP-sized structures are precipitation, emulsion, and core-shell polymerization [17]. For the preparation of MIP surfaces as membranes or electrodes, grafting can be done [11]. Better performance is normally provided by surface imprinting strategies owing to the improved access of the binding sites, favorable binding kinetics, and better reproducibility [11, 18]. Table 1 shows an overview of MIP preparation methods along with the corresponding polymerizing agents, as well as imaging moiety and application. In precipitation polymerization, polymeric microspheres are formed by the self-crosslinking of monomers/cross-linkers. The monomer and the initiator are completely soluble, but the formed polymer is insoluble in the selected solvent, thus resulting in a precipitate. The advantages of this method are the simplicity of preparation and sufficient control of product morphology [11, 19]. Emulsion polymerization is a powerful method that enables a high yield of monodispersed MIP particles. During polymerization, a monomer is dispersed or emulsified in a surfactant solution (such as sodium dodecyl sulfate). The excess surfactant creates micelles in water, and the monomer diffuses to the micelle. The polymerization is initiated by introducing an initiator molecule. The presence of water and the surface-active reagents can interrupt the hydrogen bonds between most templates and functional monomers. The synthesized MIP NPs may thus have lower template affinity and selectivity than the MIP NPs obtained by other methods of fabrication [18]. Grafting is another method for creating MIP@shell particles. In this approach, a hydrophilic polymeric layer is coated onto the surface of the MIP core. In “grafting onto” methods, reactive preformed polymers are covalently attached to the substrate and polymerized as side

chains. “Grafting from” methods involve the introduction of polymerization-initiating groups onto the NP surface from which graft chains are grown. [11] The advantages of this method are improved affinity interactions because of faster mass transfer and better control on polymer shape and morphology [18]. 2.2. Initiation methods In general, the polymerization process involves three steps: initiation, chain propagation, and termination. Most free radical polymerization methods in MIP preparation are initiated by heat, photo-irradiation, and, occasionally, redox reaction. UV irradiation usually leads to rapid initiation, but propagation of this polymer is usually slower than that of a thermochemicalinitiated polymer [18]. A specific application of the photo-irradiation initiation method was performed by Haupt’s group [20-22]. The polymer coating on quantum dots (QDs) NPs was performed by photopolymerization using the particles as individual internal light sources. After excitation of the NPs with a UV lamp (365 nm), the QDs emitted fluorescence in the green (550 nm) or the red (660 nm) region of the visible light spectrum, which overlapped with the absorption wavelength of the initiator. The local photopolymerization resulted in the formation of a thin polymer shell on the surface of the QDs [21, 22]. Similarly, carbon dots were used for localized photopolymerization in Demir et al. [20]. Electroactive functional monomers were used in Lautner et al. for the preparation of surfaceimprinted microbands on SPR chips performed by potentiostatic electropolymerization [23]. Electropolymerized MIPs have attracted considerable interest in the development of chemical sensors and biosensor thanks to their electronic properties (magnetic, conducting, and optical), similar to those of metals [24]. The inherent advantages of the electrodeposition

method are well-controlled polymer nucleation and growth and controllable film thickness and morphology [24]. Another approach for polymer coating is polydopamine (PDA). PDA can be deposited on virtually any type of substrate by oxidative self-polymerization of dopamine at basic pH without the need for crosslinking and initiating agents or organic solvents [25]. This method was employed by Chen et al. to deposit a thin imprinted film over magnetic Fe3O4/Fe nanorods [26]. In most classical radical polymerization processes, radicals are generated continuously, resulting in slow initiation accompanied by fast chain propagation. Because of the relatively high concentration of radicals, polymerization cannot be fully controlled. Consequently, this characteristic of radical polymerization leads to a very heterogeneous mixture of polymer chains of varying lengths with broad distribution of molecular weight and after crosslinking an inhomogeneous network structure [18]. On the other hand, reversible addition-fragmentation chain transfer (RAFT) is one of the subtypes of controlled radical polymerization that has been used in the polymer imprinting strategy in the works of Zhang et al., El-Schich et al., and Shinde et al. [8, 27, 28]. Here, a chain transfer agent (RAFT agent) is used to control the generated molecular weight and polydispersity during polymerization. RAFT polymerization can be used to create polymers with complex architectures such as linear block copolymers and functional polymers [29]. 2.3. Template imprinting methods As a main prerequisite, the ideal template should (1) contain functional groups that do not prevent polymerization, (2) exhibit good chemical stability during the polymerization reaction, and (3) contain functional groups that can form complexes with functional monomers [11]. The template molecule can be then imprinted as a whole, which has been

extensively studied particularly at small targets. The accuracy when imprinting the entire protein is generally satisfactory but can lead to issues with cross-reactivity and reduced selectivity in the presence of smaller polypeptides, e.g., in complex media. Whole-template imprinting of larger structures including proteins, living cells, and microorganisms can be challenging because of their dimensional structure and surface complexity. Maintaining their conformational stability during the polymerization process is a great challenge [30]. Alternatively, only a small part or a fragment of the macromolecule (a peptide or a sugar) is imprinted, also named as the epitope approach. In such an approach, similarities with the concept of antigen–antibody interactions can be found. An antibody of the immune system binds a specific antigen without the need for recognition of the entire molecule but rather a small part of it, the epitope [31]. In the epitope approach, both linear epitopes and nonlinear (conformational) epitopes can be used. Linear epitopes persist after protein denaturation or in small peptide fragments, whereas conformational epitopes are present only in folded proteins or fragments [32]. Linear epitope imprinting currently predominates. In a similar manner, MIP bioimaging applications often involve the use of structural epitopes such as glycans of the cell membrane as illustrated in Fig. 1. As a vivid example of the epitope approach, boronate affinity glycan-oriented surface imprinting has been proposed as a new strategy for producing lectin-like MIPs that can specifically recognize characteristic fragments of glycoproteins. This approach was utilized by Ge’s and Liu’s group [26, 33-35]. Glycan templates are immobilized onto NPs functionalized with boronic acid through boronate-affinity interactions. After that, a polymeric layer of appropriate thickness is formed to cover the glycans. Then, the templates are removed by washing with an acidic solution to disrupt the boronate affinity interaction [36].

Although the epitope approach is characterized by longer preparation than whole-template imprinting, epitopes can be costly and/or difficult to synthesize, and it allows achievement of higher selectivity toward the template molecule. Concerning the design of the epitope, attention should be paid to their 3D structure similar to those of antibodies, as it can significantly improve the recognition.

3. Imaging moieties The accurate and early diagnosis of diseases such as cancer is extremely important because it greatly increases the chances for successful treatment. In addition to molecular diagnostics, imaging methods are an indispensable tool for combating this severe disease. With extensive progression of current imaging technologies, fluorescence imaging is gaining popularity among research communities and multimodal (e.g., fluorescence/magnetic resonance imaging [MRI]) imaging approaches are also being developed [37-42]. When compared with other medical imaging modalities, fluorescence imaging has several benefits including lower operating costs, superior resolution, and number of detectable molecules. Organic fluorescent probes were generally used in imaging techniques [43-46]. Nonetheless, the field of nanotechnology has enabled to investigate new avenues for cancer prevention, diagnosis, and treatment. During the last decade, QDs have been in the center of great research interest because of their exceptional potential in this area. Unique properties of QDs such as size-tunable emission and versatile surface chemistry have enabled their use as a platform for tumor targeting applications [47, 48]. As the autofluorescence of biomolecules present in tissues, for example, collagen and elastin, tends to emit most of the light within the blue–green spectral range, shifting the emission of QDs toward the red and the near-infrared (NIR) range yields improved distinction of QDs for in vivo fluorescence imaging [49].

In the field of MIP-based imaging, choice of probes that enable high detection efficiency is extremely important. To address that, biocompatible semiconductor QDs [21, 22, 26], organic dye-doped polymers or silica NPs [22, 27, 28, 50-54], carbon nanodots [20], conjugated polymers (CPs) [55, 56], and alternative materials for non-luminescent imaging modalities have been used, such as gold NPs [8, 35], silver NPs [34], NPs enriched by radioisotopes [52], and iron oxide NPs [26]. Altogether, the above-mentioned applications have in common a NP-sized structure, which enables making them perfect in their size, exploiting a high surfaceto-volume ratio and malleable surface properties for active targeting (e.g., of cancer cells). We limit this chapter to the overview of the probes applied in the design of MIP for imaging, such as those shown in Fig. 3; however, further improvements are also mentioned. Review articles on general probe design and synthesis as well as development of multimodal NPs can be found elsewhere [57-62].

3.1. Luminescence imaging One of the strategies on how to incorporate imaging moieties into the polymer matrix is to add a polymerizable dye derivate to the prepolymerization mixture. For example, polymerizable rhodamine B derivative was employed by Haupt’s group [22, 50]. To obtain NPs with strong green fluorescence, fluorescein isothiocyanate (FITC) was incorporated directly into the shell of Fe3O4@SiO2 NPs in Asadi et al. [51]. Additionally, gold nanorods were encapsulated before imprinting using tetraethyl orthosilicate in the presence of FITC or an NIR797 dye in the work of Yin et al. [35]. As another example of use, Zhang et al. employed labeling of the template with FITC to validate the cellular uptake of gold MIP NPs (originally to be used for Raman imaging) by confocal laser scanning microscopy [8]. However, the fluorescence signal of FITC was not very strong in this application because of the quenching effect of gold nanorods. In the same manner, Hoshino et al. applied a

fluorescent dye (Cy5) for the labeling of melittin and fluorescent MIPs were prepared by fluorescein o-acrylate copolymerization with MIP NPs [52]. On the other hand, Wang et al. first synthesized FITC-doped silica NPs as a fluorescent core, which were further functionalized by the boronate affinity imprinting approach [33]. The photostability test proved FITC-doped silica NPs to have better photostability than free FITC molecules. Comparable synthesis of dye-enriched silica NPs was performed by Yang et al. utilizing Rubpy dye, which resulted in higher stability because of the protection provided by the silica matrix that reduced oxygen quenching of the fluorescent molecule [63]. In contrast to organic dyes, monosaccharide-imprinted QDs were used as a fluorescence signal reporter in the research of Chen et al. [26]. CdTe QDs with three different fluorescence emission wavelengths were synthesized and used for multiplexed imaging. Green- and redemitting InP/ZnS QDs were employed by Panagiotopoulou et al. [21], which are considered as less toxic than cadmium-based QDs [64, 65]. As an alternative to QDs, carbon nanodots were used by Demir et al. [20]. This type of luminescent nanomaterials in combination with a thin MIP layer was applied for the first time for biotargeting and bioimaging. Zhang et al. synthesized a polymer with two-photon fluorescence properties and specific recognition ability to sialic acid and used for monitoring sialylated glycan levels in vivo [56]. In combination with MIP, two-photon fluorescence imaging was applied for the first time. Two-photon fluorescence imaging has attracted interest in the past years because of specific properties such as high spatial resolution, increased penetration depth, and low specimen photodamage [66, 67]. Thus, this fluorescent probe not only can reduce the background interference but can also provide information of sialylated glycans at different depths in vivo. However, specific conditions to construct a powerful two-photon fluorescent probe must be met. Two-photon emitting units should have a large absorption cross section. To solve this, a CP was chosen as a fluorescent reporter [68]. The remarkable merits of the CP-based sensor

include the ability of optical amplification of an analyte binding event [68, 69]. Furthermore, two-photon absorption cross section is at CPs much larger in contrast to their small-molecule counterparts. Importantly, two-photon properties can reduce the background interference and enable high accuracy in not only target labeling but also in photodynamic therapy [68]. A fluorescent CP was also used as an MIP-based fluorescent probe for biosensing and bioimaging in the work of Liu et al. [55]. The conjugate was composed of blue photoluminescent fluorene with a benzothiadiazole unit, which has the ability to shift the fluorescence emission peak toward longer wavelengths. Fluorescent CPs have certain unique features such as light harvesting and strong fluorescence properties, low toxicity, ease of preparation, and good biocompatibility [70-75]. Interestingly, although the aforementioned downconversion materials were used as imaging agents, to the extent of our knowledge, upconversion nanoparticles (UCNPs) have not been applied in MIP-based imaging. During the past decades, the upconversion properties of NPs were highlighted showing benefits over traditional fluorescent probes. Specifically, significantly reduced autofluorescence, improved tissue penetration depth, and enhanced photostability are the inconsiderable advantages of these NPs [76]. UCNPs are rare-earth element-doped NPs exhibiting anti-Stokes behavior; thus, they can be excited by NIR light and emit light of shorter wavelengths depending on the type of lanthanide dopants used [76, 77]. In the NIR area, biological tissues have minimal light absorption, and therefore, increased penetration depth could be achieved at the tumor site [78]. In comparison with other antiStokes processes, e.g., two-photon excitation, UCNPs can be excited by a significantly lower excitation intensity of the laser [79]. UCNPs are stated to be perfect agents for high-sensitivity detection, as they allow for long-term monitoring and are fairly biocompatible and cell permeable (if small enough) [80, 81]. As a promising sign of advancement, numerous sensors for diagnostics based on inorganic UCNPs combined with imprinting technology were

presented during the past years [82-89]. Together with the development of UCNPs based on organic dyes, triplet-triplet annihilation (TTA) UCNPs [90], evidence paves the way toward the future development of MIPs for imaging. 3.2. Alternative materials for imaging The term “imaging” refers to not only fluorescence or luminescence imaging but practically any scanning method yielding a 2-dimensional picture [91]. Therefore, images can additionally be a result of diverse methods such as infrared and Raman spectroscopy [92], MRI, imaging with radionuclides, computed tomography imaging, positron emission tomography, electrochemical imaging using rastering electrodes [93], or even sophisticated scanning methods (ideally with pseudocoloring) such as laser ablation ICP-MS [94] and MALDI-MS [95]. From this perspective, an alternative readout strategy was published by Qian et al., who applied surface-enhanced Raman scattering (SERS) for cell imaging and cancer detection [96]. Although MIP NPs have been combined with SERS previously [97], advances in nanomaterials allowing for better control of the quality and physical properties now provide more reliable ways to construct SERS imaging platforms. SERS-based imaging provides numerous benefits such as high sensitivity, good biocompatibility, and the ability to multiplex [98]. The first application of SERS nanotags for selective MIP-based detection and imaging cancer cells and tissues was reported in Yin et al. [34], where Raman-active silver NPs (AgNPs) were prepared as a signal-reporting core. Sialic acid was then used as a template imprinted into a thin shell through the boronate affinity-oriented surface imprinting approach. Additionally, Zhang et al. applied gold nanorods (AuNRs) for in situ Raman detection and imaging within living cells targeting the epidermal growth factor receptor [8]. Furthermore, MIPs were prepared using 14C radioisotope-labeled acrylamide in [52]. 14C MIP NPs were used in a distribution study in the mice organs after termination. Radioactivity was determined with a scintillation counter. Moreover, [26] developed catalase-imprinted

Fe3O4/Fe@F-SiO2/PDA NPs. These NPs have shown good ability for magnetic targeting and suitability for MRI both in T1- and T2-weighted sequences and therefore enable a possibility of precise in vivo visualization of the therapeutics.

4. Applications The research of Hawkins et al. can be considered as a pioneering work in the field of MIPbased imaging [54]. FITC-albumin was employed as the protein template molecule in an aqueous phase MIP, named as the HydroMIP strategy. This was the first work reported, where a fluorescently labeled template was used and analyzed using a confocal microscope. The same group also reported a cryo sample preparation method that allows the visualization of protein-specific cavities for a polyacrylamide HydroMIP engineered for the template molecule bovine hemoglobin. These methods enable visualizing the HydroMIP in its native form with protein entrapped in the matrix, tracking the elution, and rebinding of the analyte. These findings also contributed toward the understanding of molecular imprinting protocols as well as successful in situ imaging of real-time protein denaturation events. [99] Since these proof-of-concept realizations, several research groups also focused on clinically relevant templates, which finally led to cell targeting and bioimaging by MIP NPs. Most of these targeted sialic acid [27, 28, 34, 56, 96], and other saccharides such as mannose and fucose [33] were also addressed. MIPs were able to differentiate between normal and cancer cells, with the latter presenting more of these monosaccharides on their surface [20]. In parallel, MIPs templated with glucuronic acid, a substructure (epitope) of hyaluronan, were successfully applied for biolabeling and bioimaging of intracellular and extracellular hyaluronan on human skin tissues and cells [21, 22, 50].

Not only were the applications of MIP-based imaging researched, but also hybrid approaches, such as drug delivery together with imaging, or theranostic applications can be found in the researchers’ efforts. 4.1. Molecular targets Altered glycosylation can be considered a universal hallmark of cancer cells. Particularly, sialic acid (SA) [100, 101] and fucose (Fuc) [102, 103] are overexpressed on the cell surface of most cancer cells, while mannose (Man) is overexpressed particularly in liver cancer cells [104, 105]. Aberrant expression of these monosaccharides can thus be considered as candidate biomarkers of cancer cells. Cell surface glycans are involved in cellular communication, cell development, and cell differentiation [106]. The abnormal expression of SA occurs on metastatic cancer cells, and thus, it is considered an indicator for some cancers [100, 101, 107, 108]. The phenylboronic acid (PBA) group is often used to covalently bind the molecule to cis-diol-containing compounds such as glucose by pH-dependent reversible esterification [109-111]. The limited availability of specific antibodies against SA leads to labeling of SA by MIP-coated NPs. In the work of El-Schich et al. [27], four different chronic lymphocytic leukemia (CLL) cell lines were analyzed by flow cytometry and fluorescence microscopy. SA expression was verified with lectin-FITC. SA-MIP can be used as a plastic antibody and for screening of different tumor cells at various stages, including CLL cells. The manufacturing strategy was presented previously by the same group in Shinde et al. [28]. The imprinting strategy involved RAFT polymerization of an SA-imprinted shell on silica core particles (SiO2). The polymeric shell of the NPs was equipped with guest-responsive nitrobenzoxadizole fluorescent reporter groups for environmentally sensitive fluorescence detection. The selectivity of the staining was verified by the competitor glucuronic acid. The binding of monosaccharides to SAimprinted and nonimprinted microparticles based on HPLC experiment showed strong

preference for SA, with 84% for SA-imprinted; however, the nonspecific adsorption on nonimprinted particles was relatively high (34%). In the work of Liu et al., the fluorescent CP poly(fluorene-alt-benzothiadiazole) (PFBT) was modified with PBA groups as binding sites for SA molecules [55]. SA was then easily removed from the surface of polymeric NPs by adjusting pH, followed by dialysis. The SAimprinted NPs showed a stronger fluorescence than the unmodified nanoparticles. The SAimprinted MIP NPs were able to bind to SA overexpressed in DU 145 cancer cells and did not bind to HeLa cells, while the unmodified NPs were not able to discriminate between these two cell types. The resulting SA-imprinted NPs have low enough toxicity and satisfactory biocompatibility for use as a fluorescent probe in live cell imaging. The principle of the preparation and mechanism of the nanoconstruct selectivity toward cancer cells is shown in Fig. 4. In Zhang et al., SA was again used as a target , with recognition ability toward sialylated glycan levels and combined with two-photon fluorescence detection method [56].

Fluorescence imaging of human hepatocellular carcinoma cells (HepG-2) as compared to normal hepatic cells (L02) and mammary cancer cells (MCF-7) as compared to normal mammary epithelial cells (MCF-10A) by FITC-doped silica NPs was performed in the study of Wang et al. [33]. The principle of targeting, as can be seen in Fig. 5, was based on the monosaccharide-imprinted layer, with SA, Fuc, and Man selected as templates individually. The specificity of the monosaccharide-imprinted NPs toward the imprinted template was investigated. Cross-reactivity toward nontarget molecules was less than 7.1%, 26.2%, and 22.2% for SA-, Man-, and Fuc-imprinted NPs, respectively. Man and Fuc exhibited larger cross-reactivity because of structural similarity. The monosaccharide-imprinted NPs were able to differentiate between cancer cells and normal cells. On top of that, fluorescence

intensity signals reflected the expression on different cancer cell types; thus, the application of fluorescence imaging of cancer cells was well demonstrated, and the authors believe that MIP-NPs could be extended to pathological investigation [33]. A strategy of pattern recognition of cells with the use of monosaccharide-imprinted QDs, as shown in Fig. 5, was further applied by the same group in the study of Chen et al. [26]. To measure hyaluronan, another clinically relevant biomarker of liver fibrosis and cirrhosis in chronic liver disease [112], plastic antibody for targeting its terminal oligosaccharidic subunit glucuronic acid (GlcA), was published for the first time by Kunath et al. [50]. An epitope approach of MIP fabrication was thus selected and synthesized in the form of dye-labeled NPs. GlcA-MIPs were used to image the hyaluronan on human keratinocytes and on adult skin specimens by epifluorescence and confocal fluorescence microscopy. Polymers were synthesized according to the group’s earlier work [113], where (N-acrylamido)benzamidine (AAB) and methacrylamide (MAM) were used as functional monomers. To enable optical imaging of the MIP, a polymerizable rhodamine derivative was present in the prepolymerization mixture. A reference method for hyaluornic acid localization and quantification was applied using a biotinylated hyaluronic acid-binding protein adapting the same immunostaining protocol as in the case of MIPs. Targeting both D-glucuronic acid and N-acetylneuraminic acid (NANA), the most common member of SA, was investigated in another publication by Panagiotopoulou et al. [22]. Several types of MIP-based NPs were prepared with imprinted GlcA and NANA: polymeric NPs with incorporated polymerizable rhodamine B; a MIP shell was synthesized over green and red InP/ZnS QDs with polymeric shell. Therefore, simultaneous dual-color imaging of the cells with two MIP-coated QDs was demonstrated (Fig. 6) and the nanomaterial was applied

for bioimaging of fixed and living human keratinocytes to localize hyaluronan and sialylation sites. A similar approach was described earlier by the same group [21]. As an alternative to these materials, carbon nanodots were investigated in the study by Demir et al. [20]. CDs, obtained by hydrothermal synthesis of starch and L-tryptophan, were templated with GlcA and applied for targeting of hyaluronan in human cervix adenocarcinoma cells (HeLa) and human keratinocytes (HaCaT) as models for transformed and healthy cells, respectively. 4.2. MIP-based imaging and therapy Tuwahatu et al. reviewed applications of MIP-based drug delivery systems [114]. MIPs in target delivery are understood as a subset of the 3-dimensional structure that contains memory cavities or sites, which can be used as an on-loading and off-loading domain for a variety of molecular entities. Further, Lulinski et al. [115] provided an overview of the imprinting processes and concisely described the drug release mechanism from the imprinted materials with particular attention paid to MIP drug delivery devices for ocular, dermal, intravenous, and oral routes of administration. In brief, this innovative type of pharmacotherapeutics is considered promising because it provides improved delivery profiles and/or longer release times and very often deliver the drugs in the feedback-regulated way such as pH-triggered design. Lulinski also revealed limitations that hamper the introduction of MIPs to pharmacotherapy and prevent this class of polymers from commercialization. Although numerous studies demonstrate the importance of this targeting strategy, molecular imprinting is perhaps controversial as a name for this approach. Target molecules are imprinted in the polymer; however, recognition ability of the MIPs, also described in literature as “plastic antibodies,” is in most cases not exploited here. Instead, active targeting is employed by conjugation with additional targeting molecules, or passive targeting that is based on the enhanced permeability and retention effect mediated targeting. However, the application of

molecularly imprinted particles may be used for further improvement in the form of Trojan horse-like particles, which would be imprinted toward a molecule or a cell receptor abundant at the pathological site and then simply taken up by the cells [116]. As one of the first applications of MIP-based imaging and therapy hybrid approach, Asadi et al. developed a biodegradable cross-linker agent based on fructose that was reported for the fabrication of brain-targeted MIP [51]. A magnetic Fe3O4 multicore shell structure was prepared by coprecipitation polymerization in the presence of olanzapine as the template, an antipsychotic drug, and fructose with double acts as the monomer and the cross-linker. The authors report that the magnetic field facilitates the aggregation of the carrier near the target tissue. Moreover, fructose produced during the degradation of MIP can be used as a source of energy for brain cells when there is high concentration of fructose relative to glucose [117]. The imaging functionality was obtained by FITC loaded in mesoporous silica shell created on the surface of Fe3O4 NPs. A therapeutic approach based on photothermal therapy (PTT) combined with MIPs was used for the first time in Yin et al. [35]. As a proof-of-concept, gold nanorods (AuNRs) were used as the core plasmonic nanomaterial, and SA was employed as the template by a facile boronate affinity-oriented surface-imprinting approach as demonstrated in Fig. 7. Specifically, the AuNRs with specificity toward SA were accumulated at the tumor. An NIR laser beam (750 nm) was applied to the tumor for a certain time. The SA-imprinted AuNRs bound to the tumor absorb the photon energy of the light and convert it into heat. Such heat then leads to tumor ablation within several PTT treatments, leaving the healthy tissue undamaged. In future practical applications, NIR797 dye-doped SA-imprinted AuNRs have great potential for imaging-guided targeted PTT of cancer.

Another possibility of the use of MIPs is immobilization and removal of pathogens from the bloodstream or specific molecules in a pathological site to restore the physiological conditions [116]. Evidence of the neutralization of toxic substances by MIP-NPs was published by Hoshino et al. [52]. The authors addressed the introduction of foreign substances in the bloodstream and the formation of protein coronas on the surface of the material, which can alter and/or suppress the intended function of the NPs. These complications can arise from an immunogenic response to the material. MIP-NPs with designed affinity for melittin, a cytolytic peptide present in bee venom, were introduced in the bloodstream. Melittin was labeled with a fluorescent dye (Cy5), and MIP NPs were labeled with a radioisotope 14C or a fluorescent dye (fluorescein) by copolymerization to observe the distribution of melittin and NPs in vivo in mice. MIP NPs were able to neutralize the cytotoxic peptide melittin in the bloodstream. A similar idea was tested in Ekpenyong-Akiba et al., who demonstrated the feasibility of MIP NPs targeted against senescence membrane markers to identify and clear senescent cells, which are observed in pre-malignant stages of tumors [118]. The authors characterized and validated a group of new membrane markers of senescence and used one of them, B2M, to detect senescent cells in vitro and in vivo. This work provided a proof-of-concept assessment of MIP NPs loaded with drugs that can specifically kill senescent cells. Preparation of MIPs employed covalent immobilization of the template, B2M peptide, on a solid support (glass beads). The beads were placed in the monomer mixture, which also contained the drug to be incorporated as a secondary template in the solution. The drug was not covalently nor physically linked to the NPs but only through weak interactions with the polymer and was expected to diffuse outside of the MIP NPs because of the concentration gradient. The MIPmediated cell recognition construct also showed no toxicity. To characterize their

accumulation in vivo, MIP NPs were tagged with DyLight 800 NHS Ester and Alexa Fluor 647. As expected, the results demonstrated that a wavelength of 800 nm gives a higher signal because the emitted light is less absorbed by the tissue. The wavelength of 647 nm provided lower sensitivity, and it was more difficult to separate the fluorophore signal from the background signal by spectral unmixing. From a different perspective, an integrated approach toward the application of MIP-based therapy was employed by Chen et al. [26]. The group reported a multifunctional nanoplatform for targeted therapy of cancer. The nanoplatform was composed of a magnetic Fe3O4/Fe nanorod core enwrapped by a catalase (CAT)-imprinted fibrous SiO2/PDA shell (FSiO2/PDA). Here, the CAT-imprinted NPs were able to selectively absorb CAT (399 mg·g−1 for MIP, compared to 54 mg·g−1 for NIP). This strategy was selected based on evidence that catalase protects the cell against oxidative stress, as it catalyzes the decomposition of excess H2O2 to nontoxic water and oxygen. Thus, inhibition of the bioactivity of CAT will burst levels of H2O2 and eventually induce apoptosis, which was further verified by CAT-MIP in in vitro experiments on tumor cells. Furthermore, the Fe3O4/Fe nanorod core is able to catalyze the homolysis of H2O2 in an acidic intracellular environment of tumor cells, similarly to the Fenton reagent, which yields in a large amount of ·OH radicals and triggers the apoptosis of tumor cells. Combined with the NIR light photothermal effect, CAT-imprinted Fe3O4/Fe@FSiO2/PDA NPs were able to effectively kill MCF-7, HeLa, and 293T tumor cells but were not toxic to nontumor cells. Moreover, these NPs have shown good ability for magnetic targeting and suitability for MRI. Therefore, the study confirmed that the CAT-imprinted Fe3O4/Fe@F-SiO2/PDA NPs can be practically used for in vivo imaging as a new option for visualization of nonchemotherapy for cancer.

4.3. Pseudoimmunoassays Although not considered as a bioimaging technique in the true sense of the word, several strategies entitled as MIP-based imaging have been published. A category of sensors and pseudoimmunoassays based on MIP utilizing charge-coupled device (CCD) cameras for image acquisition or SPR was reported. Chemiluminescence (CL) imaging enzyme-linked immunosorbent assay with the use of an imprinted polymer as the recognition element instead of an antibody was used in Surugiu et al. [119]. The imaging assay was developed for an antigen, 2,4-dichlorophenoxyacetic acid (2,4-D), labeled with tobacco peroxidase, and the chemiluminescence reaction of luminol was used for detection. 2,4-D-imprinted polymeric microspheres were coated at the bottom of microtiter plates. Light emission was measured in a high-throughput imaging format with a CCD camera in a competitive mode, where analyte-peroxidase conjugate was incubated with the free analyte, after which the bound fraction of the conjugate was quantified. A group of Wang et al. developed a CL imaging sensor for trans-resveratrol [120] and later also for dipyridamole [121], [122] and chiral recognition of dansyl-phenylalanine [122]. Another report of CL imaging with the use of imprinted microspheres was presented in Li et al. [123]. This work enables the measurement and high-throughput screening of ethopabate (ETP). ETP is an anticoccidial drug, and presence of ETP residues at low concentration levels in chicken tissue samples is very often controlled by law. Here, the chicken muscle samples were cut into pieces and homogenized and spiked with ETP in relevant concentrations, and extraction was then performed. Microtiter plates were coated with ETP-imprinted polymeric microspheres. The amount of bound ETP was quantified using an imidazole-catalyzed peroxyoxalate (CL) system and determined with a CCD camera.

From a different point of view, MIPs were used as a platform in SPR imaging for protein recognition [23]. The surface-imprinted polymers (SIPs) were prepared on SPR chips using photolithographic technology. Electrochemical oxidation was done to form surface-imprinted poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate), which is a material with low nonspecific protein adsorption. Molecular imprints of avidin were created. Avidinfluorescein isothiocyanate (Av-FITC) was used for the evaluation of the performance of the micropatterned chips. This new concept of the label-free optical detection technique showed outstanding sensitivity with potential to outperform that of fluorescence imaging. An MIP-based imaging system was also developed for monitoring ligand–receptor interaction [63]. An MIP receptor film was created over silica NPs with incorporated Ru-bpy dye, and the affinity toward the template was studied on the test ligand L-phenylalaninamide. Practically, the method was further extended as a template-binding membrane fabricated on a glass slide in the form of a thin layer. The binding assay was used to confirm the interaction between the imprinted membrane and template-modified fluorescent silica NPs.

5. Challenges of MIPs to be addressed Antibodies are recognition units that are well established and widely used. However, their availability for specific applications such as imaging of saccharides on the surface of tumor membranes is often limited. Here, the role of MIPs is indispensable thanks to its versatility. As shown in Table 2, MIPs of different nature can be made for practically any analyte. The synthesis of MIPs has to be well optimized and tailored to the desired target, which can be demanding, as there is no universal preparation protocol that would ensure satisfactory selectivity. The MIP technology is not very well transferable among different applications, analytes, or polymers. However, once it is tuned for a specific setup, the synthesis is inexpensive and can be scaled up in automatic reactors [124]. Inconsistency in template removal techniques, assessment of the binding capacity, and nonspecific adsorption on nonimprinted polymers are other challenges of MIPs. It is obvious that diverse polymers

require different strengths of the solvent used for template removal. While self-polymerizing PDA MIPs cannot be exposed to very harsh conditions, acrylic acid polymers are more resistant. In bioapplications, the stability of the MIP nanoconstructs is often advantageous because it offers the possibility to be sterilized or reused. There is often discrepancy in the verification of template removal among different studies. While some works assess template removal by separation methods such as HPLC, which is measured indirectly as a residuum of analyte in the sample, which was not bound by MIP, some of the studies do not provide any specific procedure on how the removal of template molecules was confirmed. In imaging applications, background signal (nonspecific adsorption) should be corrected or subtracted by the resulting signal, which needs to be addressed and unified in the future. Limits of detection achieved by the use of MIPs are comparable to antibody-based detection methods [125]. Similar to antibodies, MIPs also suffer from cross-reaction with proteins and other interferents. Another disadvantage is the lower binding capacity, which can be overcome by a suitable method of preparation [126]. Generally, imprinting sites created by the epitope approach have good accessibility for the template and demonstrate fast reversible binding kinetics [127]. Additionally, application of MIPs in vivo as a sustained release drug form is also questionable knowing the fact how difficult the template removal can be. In imaging, drug delivery or theranostics, MIP NPs are washed by bloodstream or cellular fluid, and it is challenging to control washing and/or drug release. Another challenge is that MIPs themselves do not generate any signal when the analyte from the sample is bound and therefore have to be coupled with a suitable detection method. Commonly, MIP surface is created over a functional NP, e.g., with optical properties. Despite fluorescence and luminescence methods experiencing a boom, the problem with optimal penetration depth of the light emission for in vivo applications remains unsolved. Not depending on the biorecognition element used, the optical particles generally work well at the cell level when detected in vitro by microscopic techniques. However, when applied for whole-body optical imaging, the signal may not be sufficient.

As mentioned earlier, materials requiring 2-photon excitation (e.g., UCNPs or CPs) are a promising alternative because they are tuned to be excited by wavelengths in the NIR area of the spectrum, where the penetration of light in the tissue is higher than that in the visible region. Although the emission properties of UCNPs are beneficial, the phenomenon will probably have higher impact in the development of phototherapeutic approaches rather than diagnostic ones, as the maximum penetration depth of the upconverted light does not exceed 2 cm [128].

6. Conclusion and future perspectives MIPs are a promising alternative for targets, where no natural receptors are available, such as glycanes. In contrast to multistep immunostaining protocols, molecular recognition and visualization with MIPs is usually a one-step process. Moreover, multiple labels can be applied for staining, and application of MIPs together with antibody-based staining can be performed likewise. MIPs can be synthesized against various types of target molecules ranging from small molecules such as single amino acids or sugars to proteins and even whole cells. In a nutshell, we believe that MIPs as plastic antibodies have great potential for bioimaging. Eventually, drugs can be attached to the MIP nanotags as well as dual functionality of the MIP-NPs can be incorporated, commonly depending on the core used as a carrier such as superparamagnetic or plasmonic NPs. Additionally, there is only a small range of nanomaterials that have been studied in combination with MIPs for imaging applications, such as QDs, CDs, IONPs, AuNPs, AgNP, or SiO2 NPs. Surprisingly, UCNPs, both organic and inorganic, have not been applied in this field despite numerous advantages in bioimaging, such as background-free detection and resistance to photobleaching. Therefore, we believe that a broad area of possible extension of applications remains uncovered, and further advanced applications are yet to be developed.

Acknowledgment This work has been supported by the Internal Grant Agency of Mendel University in Brno (IGA MENDELU IP 023/2019); Czech Science Foundation (project no. 17-12774S); and the Ministry of Education, Youth and Sports of the Czech Republic, under the project CEITEC 2020 (LQ1601). Tereza Vaneckova is a Brno Ph.D. Talent Scholarship Holder funded by the Brno City Municipality. Conflict of Interest The authors declare no conflict of interest. Figure Captions

Fig. 1: Overview of the preparation methods of the MIP

Fig. 2: Schematic representation of glycans and glycoconjugates on the cell membrane (GlcNAc – N-acetylglucosamine, GlcA – glucuronic acid, GlcNH2 – D-glucosamine , Glc – glucose, GalNAc – N-acetylgalactosamine, Gal – galactose, Man – Mannose, Xyl – xylose, Fuc – fucose, Sia – sialic acid, IdoA – iduronic acid). Adapted and redrawn from [22].

Fig. 3: Dyes and nanoparticles generally applied in MIP-based imaging.

Fig. 4: SA-imprinted NPs, top: schematic illustration of the preparation of the MIP and mechanism of their selectivity toward cancer cells, bottom: confocal laser scanning microscopic images of DU 145 and HeLa cells incubated with (a) SA-imprinted NPs, (b) unmodified NPs, and (c) polymer/SA NPs for 24 h at 37 °C. Reprinted with permission from [55].

Fig. 5: Pattern recognition of cells by multiplexed imaging with monosaccharide-imprinted QDs. Reprinted with permission from [129].

Fig. 6: Confocal image showing multiplexed labeling of GlcA and NANA on fixed human keratinocytes by MIPGlcA-QDs (green) and MIPNANA-QDs (red), respectively. Nuclear staining with DAPI. (B) Epifluorescence image of vital keratinocytes grown on coverslips and stained with both MIPGlcA-QDs (green) and rhodamine-MIPNANA (red) (bottom) and phase contrast (top). Reprinted with permission from [22].

Fig. 7: Illustration of the targeted photothermal therapy by SA-imprinted AuNRs@SiO2. Reprinted with permission from [35].

Table 1: Overview of the imprinting strategies in MIPs applied for imaging Method of imprinting

Monomer

Crosslinker

solvent extraction/evaporatio PFBT-PBA n single-emulsion method

-

precipitation polymerization

MAA, AM, or 4VP

PETRA

re-precipitation polymerization

PFBT-PBA

-

precipitation polymerization

4-VP

TRIM

Initiator

ammonia

Solvent

THF

Imaging moiety/carri er CP with 2photon FL properties

Template

Application

SA

monitoring of sialylated glycan levels in [56] vivo with 2photon FL imaging

BPO, acetonitrile PO-CL ethopabate DMA acidic fluorescent condition THF, methanol SA CP s CL reaction of tobacco peroxidase AIBN methanol/H2O 2,4-D and luminol, polymeric microspheres

precipitation polymerization

MAA

EGDMA

AIBN

acetonitrile

PO-CL

precipitation polymerization

MAA

TRIM

AIBN

acetonitrile

PO-CL

precipitation

fructose with

fructose

AIBN

acetonitrile/D

magnetic

CL imaging

Ref.

[123]

selective cancer [55] imaging CL imaging MIP-based ELISA (competitive)

CL imaging MIP-based trans-resveratrol sensor for transresveratrol CL imaging assay for dipyridamole recognition of dipyridamole olanzapine brain drug

[119]

[120]

[121] [51]

polymerization

double acts, with double methacryloxypropy acts l trimethoxysilane

MSO

fluorescent Fe3O4@SiO2FITC NPs

GlcA

biolabeling and bioimaging of hyaluronan on [50] skin tissues and cells

GlcA, NANA green and red InP/ZnS QDs

labeling of hyaluronan and sialic acid on [22] fixed and living cells

green and red GlcA, NANA InP/ZnS QDs

multiplexed cell [21] targeting

DMSO

CDs

GlcA

targeting hyaluronan in HeLa and HaCaT cells

DMSO

Rubpy

Lstudy of ligandphenylalaninami [63] receptor affinity de

precipitation polymerization

AAB, MAM

EGDMA

ABDV

DMSO

polymerizabl e rhodamine B

precipitation polymerization

AB, MAM

EGDMA

ABDV

DMSO

polymerizabl e rhodamine B

DMSO, toluene

DMSO, toluene

shell 1: HEMA, grafting (localized EbAM, shell 2: AB, EGDMA photopolymerization) MAM shell 1: HEMA, grafting (localized EbAM; shell 2: photopolymerization) AB, MAM

EGDMA

grafting (localized AB, MAM photopolymerization)

EGDMA

grafting polymerization

EGDMA

MAA

eosin Y/TEA, irr. 365 nm eosin Y/TEA, irr. 365 nm coumarin 6/TEA, irr. 365 nm ACVA (grafting initiator)

delivery

[20]

Method of imprinting photolithography method (electrochemical deposition)

Monomer

Crosslinker

Initiator

EDOT, NaPSS

-

0.75 V for water 25 s

Fe3O4/Fe nanorods

Solvent

Imaging moiety/carrier

Template

Application

label-free

avidin

detection of proteins by SPR [23] imaging

Ref.

integrated nanoplatform of magnetic [26] targeting, MRI and cancer therapy Recognition, neutralization, [52] and clearance in vivo screening of different chronic lymphocytic [27] leukemia cell lines by FL microscopy and flow cytometry

oxidative selfpolymerization

dopamine

-

basic condition 10 mM s, O2 Tris-HCl presence

copolymerization

NIPAAm, AM, AAc, TBAm

BIS

APS, TEMED

water, ethanol

Cy5, radioisotope 14C, melittin fluorescein

RAFT-mediated grafting

4-VPBA, AEMA, NBDAE

EGDMA

ABDV

methanol

core-shell SiO2 with NBD fluorophore

SA (competitor GA)

RAFT-mediated grafting

4-VPBA, AEMA, NBDAE

EGDMA

ABDV

methanol

core-shell SiO2 with NBD fluorophore

SA (competitor GA)

labeling of cell surface glycan

[28]

-

-

N/A

AuNRs (FITC)

EGFR

Targeted live cell Raman imaging,

[8]

self-assembled monolayers using NIPAAm RAFT polymerization

catalase

boronate affinityoriented surface imprinting approach

FPBA, TEOS

-

ammonia

ethanol

FITC-doped SiO2 SA, Fuc, Man NPs

boronate affinityoriented surface imprinting approach

FPBA, TEOS

-

ammonia

ethanol

Ag/PATP@SiO2 SA NPs

boronate affinityoriented surface imprinting approach

prepolymerization FPBA, APTES; TEOS

ammonia

ethanol

AuNRs (FITC or SA NIR797 dye)

boronate affinityoriented surface imprinting approach

APBA, TEOS

-

ammonia

ethanol

CdTe QDs

Man, SA, Fuc

MBAA

APS, TEMED

water

FITC

albumin, BHb

aqueous phase molecularly imprinted AM polymer strategy

visualization of cancer biomarkers in vitro and in vivo targeting of human hepatoma and mammary cancer cells over noncarcinogeni c cells surfaceenhanced Raman scattering imaging plasmonic MIP NPs for NIRtargeted photothermal therapy of cancer pattern recognition of cancer cells by multiplexed imaging proof-ofconcept of imaging

[33]

[34]

[35]

[26]

[54]

strategy, twophoton confocal microscopy aqueous phase molecularly imprinted AM polymer strategy

MBAA

APS, TEMED

water

-

BHb

TEM imaging of all MIPs

Abbreviations

2,4-D – 2,4-dichlorophenoxyacetic acid 4-VP – 4-vinylpyridine 4-VPBA – 4-vinylphenylboronic acid AAB – (N-acrylamido)-benzamidine AAc – acrylic acid AB – (4-acrylamidophenyl)(amino)methaniminium acetate ABDV – 2,2’-azobis(2,4-dimethylvaleronitril) ACVA – 4,4'-azobis (4-cyanovaleric acid) AEMA – 2-aminoethyl methacrylate hydrochloride AIBN – 2,2'-azobisisobutyronitrile AM – acrylamide APBA – 3-aminophenylboronic acid

EbAM – N,N′ethylenebis(acrylamide) EDOT – 3,4ethylenedioxythiophene EGDMA – ethylene glycol dimethacrylate EGFR – epidermal growth factor receptor FITC – fluorescein isothiocyanate FL – fluorescence FPBA – 4-formylphenylboronic acid Fuc – fucose GA – glucuronic acid HaCaT – human keratinocytes HeLa – human cervix adenocarcinoma cells HEMA – poly(2-

NaPSS – poly(sodium 4-styrenesulfonate) NBD – nitrobenzoxadiazole NBDAE – 2-[(7-nitro-2,1,3-benzoxadiazol-4yl)amino]ethyl acrylate NIPAAm – N-isopropylacrylamide PATP – p-aminothiophenol PBA – phenylboronic acid PETRA – pentaerythritol triacrylate PFBT – poly(fluorene-alt-benzothiadiazole) PFBT-BO – poly(fluorene-alt-benzothiadiazole)boronic acid PO-CL – peroxyoxalate chemiluminescence RAFT – reversible addition-fragmentation chain transfer Rubpy – tris(2,2′-bipyridine)ruthenium(II)

[99]

APMA – N-(3-aminopropyl) methacrylamide hydrochloride APS – ammonium persulfate APTES – (3-aminopropyl)triethoxysilane BHb – bovine hemoglobin BPO – benzoyl peroxide CDs – carbon nanodots CP – conjugated polymer DMA – N,N-dimethylaniline DMSO – dimethyl sulfoxide

hydroxyethylmethacrylate)

hexafluorophosphate

irr. – irradiation MAA – methacrylic acid MAM – methacrylamide Man – mannose MBAA – N,N'methylenebisacrylamide MRI – magnetic resonance imaging N/A – not available NANA – N-acetylneuraminic acid

SA – sialic acid SiO2 – silica shell SPR – surface plasmon resonance TBAm – N-tert-butylacrylamide TEA – triethylamine TEMED – N,N,N',N'-tetramethylethylenediamine TEOS – tetraethyl orthosilicate TRIM – trimethylolpropane trimethacrylate

Table 2: Characteristics of MIPs in comparison with antibodies Advantages of MIPs

Disadvantages of MIPs

Stability

No universal preparation protocol

Economically efficient

Inconsistency in template removal

Versatility (can be prepared for any analyte)

Detection methods

Do not require work with animals

Lower binding capacity

Possibility of large-scale production

Cross-reactivity

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