Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-021-03313-8 REVIEW Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues Anna Herrera-Chacón 1 & Xavier Cetó 1 & Manel del Valle 1 Received: 18 February 2021 / Revised: 18 March 2021 / Accepted: 24 March 2021 # Springer-Verlag GmbH Germany, part of Springer Nature 2021 Abstract Molecularly imprinted polymers (MIPs) are artificially synthesized materials to mimic the molecular recognition process of biological macromolecules such as substrate-enzyme or antigen-antibody. The combination of these biomimetic materials with electrochemical techniques has allowed the development of advanced sensing devices, which significantly improve the perfor- mance of bare or catalyst-modified sensors, being able to unleash new applications. However, despite the high selectivity that MIPs exhibit, those can still show some cross-response towards other compounds, especially with chemically analogous (bio)- molecules. Thus, the combination of MIPs with chemometric methods opens the room for the development of what could be considered a new type of electronic tongues, i.e. sensor array systems, based on its usage. In this direction, this review provides an overview of the more common synthetic approaches, as well as the strategies that can be used to achieve the integration of MIPs and electrochemical sensors, followed by some recent examples over different areas in order to illustrate the potential of such combination in very diverse applications. Keywords Molecularly imprinted polymers (MIPs) . Voltammetric sensors . Electronic tongue . Molecular imprinting . Polymerization . Immobilization Introduction methods have turned into a key element to improve its quality and to allow its traceability [3]; clinical diagnostics, where the Over the last decades, there has been an exponential increase identification of key biomarkers and their normal ranges al- in the demand for the identification, classification or quantifi- lows the detection of several pathologies [4]; etc. cation of chemical and/or biological species in more or less Beyond the challenge that the analysis of such compounds complex matrices. Such increase is not limited to a particular can represent by itself, there is also a demand for such methods field, but a general trend of a more knowledgeable society that to be economic, fast-response, simple, portable and low-energy demands, from the stakeholders and policymakers, a better consumption. In this direction, electrochemical sensors offer fast understanding, monitoring and control of the composition of and accurate information in a cost-effective manner. Besides, daily life products. Some examples of these increases can be their high sensitivity, wide linear range, minimal power require- found in the environmental field, where the number of emerg- ment, potential for miniaturization and portability, as well as ing contaminants is growing continuously (with over 100,000 ease of operation, are some of their several advantages [5]. For chemicals registered) [1, 2]; food control, where analytic these reasons, among the incorporation of durable and reusable sensors, the field of sensing and biosensing is gaining popularity among the scientific community and is one of the most growing Published in the topical collection Analytical Applications of Biomimetic fields in recent publications. Recognition Elements with guest editors Maria C. Moreno-Bondi and Elena Benito-Peña. In order to improve the performance and selectivity of sen- sors, many different materials and (bio)compounds have been * Manel del Valle used when developing electrochemical sensors [6, 7]. On the manel.delvalle@uab.cat one side, there is the usage of (bio)catalysts (such as enzymes) that specifically catalyse the conversion of the target analyte to 1 Sensors and Biosensors Group, Department of Chemistry, another product more easily detectable, or electrocatalysts that Universitat Autònoma de Barcelona, Edifici Cn, 08193 Bellaterra, Barcelona, Spain act as mediators for a more efficient electron exchange and/or Herrera-Chacón A. et al. faster reaction rate, which results in, e.g. a lower oxidation/ previously mentioned complex. Upon synthesis of the poly- reduction potential and/or increase in the sensitivity [8]. In this mer, the next step is the removal of the template molecule, in sense, the progress in nanoscience has led to a range of new order to yield a material able to selectively rebind to the target nano-structured materials that have emerged as an alternative analyte with high specificity even in the presence of structur- to the respective bulk materials given its higher surface/mass ally analogue interfering compounds. Non-imprinted poly- ratio and improved electrochemical properties [9]. On the oth- mers (NIPs) are synthesised in the same manner, but without er side, there is the usage of (bio)chemical recognition ele- the template presence for comparison purposes. As schema- ments that do not affect the electrochemical responses direct- tized in Fig. 1, in the case of MIP synthesis, the monomer(s) ly, but enhance the selectivity of the developed sensors by are first spontaneously arranged around the template to obtain specifically binding to the desired analyte (e.g. affinity biosen- a tailor-made cavity complementary to it. If we consider the sors, which include immunosensors and genosensors) [10]. NIP synthesis, there is no arrangement process due to the Despite the high performance of such type of biosensors, absence of the template; producing a polymer without imprint- those present some drawbacks such as its cost, long-term sta- ing (against the template molecule) and therefore a less func- bility or instability out of the physiological conditions, be- tional cavity. Reason for which the NIP polymer is used as a tween others. In this direction, to spawn new application control of the synthesis by itself. fields, there is a necessity to obtain synthetic biomimetic rec- Finally, in order to be used for electrochemical sensing, as ognition elements that may substitute natural analogues such the synthesis of these polymers provides plastic insulator ma- as antibodies or enzymes. Advantages of working with these terials, the proper integration into chemical sensors (where the artificial receptors are not only because of its affordability but transducer will convert the binding event into an analytical also due to the possibility to work in a wide range of condi- signal) is required. This is the reason why optical applications tions (pH, temperature, solvents, etc.) or out of strict physio- are more common when employing MIPs. Despite their poor logical conditions. electrical properties, their incorporation into chemical sensors In this context, several synthetic (bio)molecules have been clearly improves the selectivity and the sensitivity of the latter, evaluated from the principles of host-guest chemistry, includ- as MIPs allow the extraction and pre-concentration of the ing the use of aptamers (aptasensors), molecularly imprinted target analyte from sample complex matrices, therefore per- polymers (MIPs), crown ethers or cyclodextrins; more recent- mitting an enrichment and posterior stripping methodology. ly, the use of metal-organic frameworks (MOFs) or covalent As these materials gain attention, new synthetic routes, po- organic frameworks (COFs) has also attracted significant at- lymerizations and immobilizations are being explored in order tention as artificial receptors. However, the poor chemical to improve the overall performance of electrochemical sensors. stability in aqueous solutions and low electrical conductivity In this direction, this manuscript aims to present and critically of the latter hinders their use in electrochemical systems [11]. review the more common approaches for the synthesis of MIPs Among the above list, aptamers and MIPs have clearly dem- as biomimetic synthetic receptors, and their incorporation into onstrated a potential and performance above the rest, but with electrochemical sensors. Finally, some of the most relevant MIPs still being a cheaper and more versatile option in terms applications recently reported in the literature over different of operation conditions given its higher stability. scenarios will also be reviewed in order to illustrate the poten- Among biomimetic artificial receptors, MIPs represent a tial of MIPs in combination with electrochemical sensors. promising alternative due to its simple synthesis, comparable performance to affinity bioreceptors, tunability, high stability and low-cost. These polymers are synthesised based on Synthesis template-induced formation of complimentary recognition cavities, which are thus made specific in shape, size and func- Molecular imprinting includes the design and synthesis of the tionality against a target chemical or biological molecule (the molecular recognition polymers and comprises a plethora of template). Indeed, the recognition of the template by the MIP possibilities. The field started in the 1930s when Polyakov has been compared to the Fischer’s lock and key analogy used reported an unusual selective behaviour of some silica parti- to describe the preferential interaction between an enzyme and cles depending on the different additives and solvents its substrate [12]. MIPs synthesis commonly starts by mixing employed during its preparation [13]. This was in the same the template with one or more functional monomer(s) in an decade that the idea of the antibody recognition was under appropriate solvent, which allows the formation and debate by Bernil [14] and Pauling [15]. Some years after- solubilisation of the pre-polymerization complex (based on wards, Dickey [16], in the same direction that Polyakov did complementary forces between chemical groups of both mol- earlier, published about the precipitation of highly selective ecules). After that, there is usually the addition of a cross- silica gel particles towards dye methyl orange with other dif- linker and a radical initiator, commonly under free oxygen ferent dyes present. But it was not until 1952 that this idea was conditions, mechanism that knits the polymer around the Nobel laureated when Martin and Synge presented their study Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues Fig. 1 Schematic representation of the synthesis of biomimetic artificial receptors: (A) molecularly imprinted polymer (MIP) and (B) non-imprinted polymer (NIP) describing the induction of selective footprints using methyl will be discussed, taking into account its suitability to be later orange as an indicator ligand during the separation of amino used for the development of electrochemical sensors. acid derivatives [17]. The former settled the basis for what Klotz [18] and Wulff [19] described as molecular imprinting Covalent imprinting when reporting the synthesis of a non-siliceous cross-linked polymer around a template molecule, and Sagiv [20] about the Covalent imprinting method was developed and first reported concept of silica imprinting. However, it was not until the by Wulff and Sarhan [19, 23], for the imprinting of D-glyceric 1990s that the interest on exploiting MIPs started to grow acid (the template) using molecules with boronic acid and exponentially, developing MIPs towards a diverse range of amine-based functionalities for the steric arrangement and templates such as ions, molecules, macromolecules, viruses, polymerizable vinyl groups combined with divinylbenzene micro-organisms or nanoparticles, among others. as cross-linker. Molecular imprinting via covalent forces was Prior to moving forward to the synthesis of MIPs, special the first method reported for the design of artificial receptors. mention has to be made to the NIP and its importance. For This method is highly suitable when a great level of precision binding comparative purposes, a NIP should be synthesised in on the specificity of the imprinting is desired due to the high parallel. The aim is to demonstrate that there is actually an stability of the template-monomer(s) complex, which arises imprinting effect on the synthesized polymer. In this manner, from the strong bonds formed between them, and that theoret- by using the NIP as a benchmark for our MIP, it is possible to ically leads to highly specific and high-affinity MIPs. evaluate the imprinting factor (IF = KMIP/KNIP, where K is the However, what is its main advantage becomes also its major affinity constant or the sensor’s response), which is a measure drawback given the difficulty to remove the template once the of the strength of the interaction of the imprinted polymer MIP is synthesized. This is evidenced in the poor recovery of towards the template molecule. Therefore, the best MIP is the template after the polymerization, but also in the increased the one that has the largest IF value, confirming a good im- difficulty in the rebinding/removal of the template when used printing, while values close to 1 indicate that the imprinting as a MIP, which leads to slower kinetics. effect is almost negligible, which means that the interaction To proceed with this approach, the template molecule re- between the MIP and the template is rather unspecific, more quires having at least one functional group suitable to be related to surface interactions than to any generated cavities. derivatized; common examples found in the literature include As previously mentioned, the imprinting technique is based alcohols, aldehydes, ketones, amines or carboxylic acids. on the formation and arrangement of the pre-polymerization After creating the tailor-made cavities, the template needs to complex between the template and the functional mono- be removed by chemically cleaving the bonds with the syn- mer(s), followed by the synthesis of the polymer matrix on thesized MIP. This can be achieved by, e.g. acidic hydrolysis its surroundings, and finally, the template removal [21, 22]. or reductive cleavage of the ester bonds with concentrated Precisely, the main difference among the different approaches acids or bases, or through the application of high pressure or to perform the synthesis is the nature of the pre- supercritical CO2, among other ways. Thus, other drawbacks polymerization binding and how the template molecule is fi- of this synthetic approach are: (1) the template modification nally removed (Fig. 2). In this direction, over the next subsec- implies an extra step, and (2) the limited number of species tions, the more common approaches for the synthesis of MIPs that may form a covalent bond with reversible properties (the will be presented and their main advantages/disadvantages list includes carboxylic and boronic esters, ketals, disulphide Herrera-Chacón A. et al. Fig. 2 Diagram of the main imprinting methods: a Covalent and semi-covalent imprinting, b metal-ion exchange imprinting and c non-covalent imprinting bonds, acetals and imines), which translates into a limitation compromised due to the heterogeneity from the different of the species that can be used as a template and, consequent- pre-polymerization complexes formed, the direct competition ly, for which a MIP can be synthesized by this strategy. from the solvent, the cross-linker (that is added in high pro- portion in order to obtain a polymer with more rigidity) or the underestimation of the chaos generated by polymerization Non-covalent imprinting process that is often promoted by thermal-induced polymeri- zation process, the so-called free radical polymerization Non-covalent imprinting is by far the most widespread meth- (FRP). odology. Mosbach’s group promoted and disseminated this approach in the 1980s decade [24–26], causing a great popu- larization of the imprinting technique and an exponential Semi-covalent imprinting growth in publications over the 1990s and 2000s [27] that seems it has not stopped yet. The nature of the pre- The semi-covalent approach seeks to combine the advantages arrangement in non-covalent imprinting (with a wider range of both the covalent and the non-covalent synthesis, while of chemical functionalities and a relatively simpler synthesis counterbalancing their disadvantages. This approach can be process) became a milestone that opened a full variety of uses attributed to Wülff [28], but it was not until a few years later and applications over several scientific areas. The types of when it was formally reported with the use of “sacrificial interactions that are predominant for the formation of the spacer” by Whitcombe et al. [29]. The main idea is to create self-assembled pre-polymerization complex in this approach a MIP based on covalent imprinting (pre-polymerization com- are the Van der Waals forces, dipole-dipole, charge-dipole, plex with covalent bonds), and after the polymerization step, cation-π, π-stacking, hydrogen bonding or electrostatic the covalent features involved are replaced by non-covalent (charge-charge), between others. Whereas for the synthesis functionalities so that the rebinding of the template is based on of the MIP, functional monounsaturated monomers such as non-covalent interactions. This mixed process thus benefits vinylic, acrylic or methacrylic monomer are used; together of the lower non-specific interactions and highly precise cav- with a high amount of cross-linker from the same nature (vi- ities of covalent imprinting, and of the more favourable tem- nylic, acrylic or methacrylic). The mechanism of choice is plate rebinding process of non-covalent imprinting (the rebind usually free radical polymerization (thermally or UV induced) is only affected by diffusion parameters, without counting any which provides the final organic network material. other kinetic parameter than diffusion). The main advantages of non-covalent imprinting are the However, crowding and steric hindrance between the MIP limitless type of templates that could be used, its ease of prep- and the template may arise due to the change in functionali- aration, the faster and more efficient template removal and, ties, which may cause a loose in the polymer binding capacity, significantly, the more favourable rebinding kinetics. hampering the imprinting. In order to overcome such draw- However, the specificity of the binding sites is clearly back, a linker group (the so-called sacrificial spacer) may be Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues used to connect the template and the monomer(s), which is added during the polymerization, achieving their incorpora- lost during the template removal. In this manner, the sacrificial tion into the polymeric matrix. Lastly, the removal of the spacer serves, first, to covalently attach the template and the template could be done by, e.g. overoxidation of the polymer functional monomer(s) during the pre-polymerization, while or the template itself, a step that is extremely fast in compar- at the same time serves to prevent steric effects upon template ison with other approaches. removal. Some examples of sacrificial spacer groups that can As already commented, a main asset of this approach is that be found in the literature include the use of carbonate ester, it consists of a one-step electro-synthesis, which allows the carbamate, urea, salicylamide or silyl ether. Nevertheless, the integration of the material onto the electrode surface with use of sacrificial spacers reduces the list of possibilities of good adherence, and that the thickness of the MIP layer can tailor-made cavities by imprinting technique, adds an extra be tuned by changing the total charge flown at the electrode, coupling step that needs to be optimised and provides a less- the polymerization media or the electrolyte nature. On the effective template recognition if the necessary attraction di- other hand, the main drawbacks are related to the more limited poles are not strong enough. choice of monomer(s) and the interactions that those might have with the template, and that the template should be stable Metal-ion exchange imprinting during the synthesis step in the potential window used for the electropolymerization to ensure that precise and specific cav- Similar to semi-covalent imprinting method, which arose ities are generated. No only this, but also the other compo- as an alternative that sought to improve the performance nents that may be added into the mixture need to not have of existing methodologies, metal-ion exchange imprinting functional groups able to polymerize to avoid creating hetero- aims to take advantage of metal ions to create ionic bonds geneous binding sites. (which are stronger than hydrogen bonds or electrostatic Despite not being considered yet a separate category for the interactions) that facilitate the interaction between the imprinting technique [32], the use of electropolymerized MIPs monomer and the template molecule, while also combine in (bio)sensing has skyrocketed over the last years. Recently, the benefits of covalent and non-covalent imprinting. That published works seem to favour in this approach due to its is, metal ions are used as an assembly pivot for the for- advantages, such as its simplicity or its integration ability. mation of the pre-polymerization complex. The metal ion Some examples include the use of polymerizable functional may form a complex between the template with mono- monomer(s) such as o-phenylenediamine (oPD), aniline, thio- mer(s) that form coordinative bonds with metals, or by phene or thiophene derivatives (Th), chitosan (Ch), acrylam- using ionophores for the imprinting. Interaction of the ide and the most widely used pyrrole (Py), alone or in metal ions and stability of the complexes formed can be derivatized forms. tuned by varying the metal, its oxidation state and/or the ligand characteristics. Moreover, apart from being used as Strategies to improve molecular imprinting a monomer through the integration of the accompanying ligand (e.g. porphyrins) into the polymeric material, they Computational approaches for the design of MIPs can also act as template for the imprinting of metal ions. This approach was first proposed by Fujii et al. for the Although it does not represent a synthetic route itself, and its imprinting of a polymer able to discriminate between the ste- usage precedes the actual synthesis of the imprinted polymer, reoisomers of an amino acid [30], demonstrating the huge we consider that special mention is deserved to the use of potential of the obtained MIP and the approach. Despite its computational methods in the design of MIPs, which may evident potential, is clear that this approach is limited to strat- facilitate further improvement of the high affinity of such egies containing complex-forming groups with metals. polymers through rational design protocols [33–36], with ev- ident reduction of experimental effort. Computational In situ electropolymerized MIP sensors methods for receptor design appeared in the late 1990s, but it was not until 2001 when Piletsky et al. demonstrated that The first electropolymerized MIP-based sensor was reported good predictions could be obtained, avoiding running large in 1999 by Malitesta et al. for the detection of glucose by sets of experiments that these methods were widely acknowl- quartz crystal microbalance (QCM) [31]. The MIP was ob- edged [37]. In this manner, screening of the functional mono- tained from a solution that contained o-phenylenediamine mer, selection of the cross-linker and the solvent, optimisation (oPD) as the electropolymerizable monomer, the template of monomer(s)-template ratio or selectivity analysis are com- and a supporting electrolyte. In the procedure, the authors putationally simulated by molecular modelling and computa- achieved both the synthesis and immobilization of the MIP tional design. To this aim, several approaches have been re- onto the electrode surface at the same time. Furthermore, a ported in the literature, including molecular mechanics/ cross-linking co-monomer or different nanomaterials may be molecular dynamics or one of the broad quantum Herrera-Chacón A. et al. mechanics-based techniques (semi-empirical, ab initio or den- surfaces to form different architectures such as core-shells, sity functional theory (DFT)). Alternatively, chemometric layers or beads. methods have also been used for the optimization of synthesis parameters [38, 39]. The advantages derived from the use of Type of polymerization such methods are the preparation of high-affinity polymers with control over their binding strength on the one side, while Among the different types of polymerization, MIPs are gen- also allowing for a significant reduction in time and resources erally produced by free radical polymerization (FRP), con- that would be required with empirical screening methods on trolled radical polymerization (CRP), photopolymerization the other side. Moreover, these methods contribute to the or electropolymerization [43]. greater understanding of monomer-template complexation From those, FRP is the most widely used strategy. The and the impact of polymerisation on the structure of the bind- reaction is conducted under mild conditions, either in bulk ing site, unveiling the production of more advanced materials or in solution, and it may be initiated by heating or UV-vis (e.g. responsive materials). irradiation. It can be used with several monomers, and from the preparation point of view, it provides a simple and rapid ap- Dummy MIPs proach. Some disadvantages are that it does not allow control over the size of the macromolecules, the irreversible ter- Although one of the advantages of MIPs is the possibility to mination reaction and the possibility of uncontrolled atom synthetize highly selective receptors for almost any transfers. (bio)compound, there is certain situations in which this is In this direction, there are other variants that could help to not as straightforward. Namely, when the target template (i) control the size, shape and molecular weight distribution such as is unstable under the conditions required to carry out the po- CRP or living radical polymerization (LRP). CRP is mainly used lymerization or its poor solubility hinders the imprinting, (ii) it in through activation-deactivation cycles, which help to control is considered a hazard or safety concerns arise from its manip- the polymer growth and to obtain polymeric chains of similar ulation, (iii) is very expensive or (iv) to solve problem of length. CRP can be used for several types of chemical species, template leakage. In such scenarios, a structural analogue and includes several strategies such as atom transfer radical poly- might be used as the template to carry out the imprinting merization (ATRP), nitroxide-mediated polymerization (NMP), instead of the target analyte; an approach that is known as iniferter-mediated polymerization or reversible addition- dummy molecular imprinting or simply referring to the fragmentation chain transfer polymerization (RAFT). As already synthetized polymer as dummy MIP (DMIP). This was first mentioned, the main advantage of CRP methods is that it yields reported by Andersson et al. [40], although the term was not monodisperse polymer particles with uniform morphology, e.g. coined until a few years later. Examples of scenarios in which as it happens with dendrimer structures. DMIPs can be considered the more attractive or effective ap- Photopolymerization is mainly employed to obtain thin proach involve the synthesis of MIPs towards compounds films with controlled thickness requirements, often in the such as TNT due to being an explosive [41] or bisphenol A nano-scale level. Its main advantages are the tight control on (BPA) due to its leakage [42]. the film thickness. However, given the low penetration of the UV radiation, there is a limit in the thickness or size of the polymer. Another limitation is that it may not be suitable for Polymerization all molecules as it might be the case where the template may degrade once exposed to UV radiation. The polymerization is a key part of the MIP synthesis process Lastly, electropolymerization approach is mainly used to as the final polymer form changes the whole functionality and obtain films directly onto the electrode surface, as already affects its main purpose, the imprinting. For example, a fast described in the “In situ electropolymerized MIP sensors” uncontrolled polymerization reaction might generate a poly- section. For obvious reasons, this option has been mostly mer with non-accessible cavities or generate a non-rigid poly- employed in the field of electrochemical sensors. Its main mer that may become useless depending on its intended ap- advantages are the relative simplicity of the polymerization plication. In this sense, a plethora of polymerization ap- process compared to the previous methods, the high reproduc- proaches can be used, from which over the next subsections ibility and control over the polymerization, or the compatibil- we will focus on the more common polymerization types and ity with aqueous media. methods. Moreover, the polymerization step can be further tuned by changing the ratio of the solvent over the other re- Method of polymerization agents, the ratio of the monomers and the template, the initi- ation method or source, the surface or place where it occurs, Checking MIP progress, by the 1990s decade, the imprinting etc. Besides, the polymer chains can be incorporated to several technique gained popularity by offering bulk materials with Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues precisely tailor-made cavities. However, the created porous its structure during the mechanical grinding, there is a risk of were not always as desired, and the size of the obtained par- altering the imprinting. Moreover, the pores are not as acces- ticles was not homogeneous enough even after grinding the sible as in other approaches, which also might hinder the mass monolith; all this produced a limited material with template transport and diffusion. For this reason, the template removal leakage and poor posterior rebinding. Especially in the elec- is more problematic, which is reflected in the very poor recov- trochemical field, this material was inadequate due to its dif- ery of the template after the polymerization. An example of ficult integration into electrochemical sensors, particularly in this synthesis can be found in the work of Urraca et al. for the the commonly preferred form: a thin layer incorporating this non-covalent imprinting of zearalenone [44], an estrogenic biomimetic polymer to provide a better and enhanced trans- mycotoxin, or for the synthesis of water-compatible MIP for duction thanks to improved diffusion. fluoroquinolone antibiotics using enrofloxacin (a fluoroquin- Due to the disadvantages that the monolith synthesis pre- olone) as the template [45]. sented, several alternative approaches were developed in the following years, advancing in new synthesis and polymeriza- tion strategies that facilitate the incorporation of these artificial Precipitation polymerization receptors into new sensing platforms. The revolution became when MIPs were synthesized as nanoparticles (NPs), offering Precipitation polymerization is a superb approach to obtain a higher-surface-to-area ratio and improving their binding ki- uniform and spherical sub-micrometre particles with the prop- netics, commonly through precipitation polymerization. er imprinting properties. Different ratios of reagents can be In this regard, MIPs prepared by FRP, CRP or LRP can be used from a highly cross-linked to an excess of solvent mix- prepared by bulk, precipitation, emulsion, core-shell, suspen- ture, which converts this approach in one of the more versa- sion, multi-step polymerization, etc., which choice will deter- tiles, and consequently, one of the most commonly used. The mine how polymer particles are obtained. Over the next sub- polymer particles grow in solution until they reach the critical sections, those will be briefly presented, while Table 1 sum- size, at which moment they precipitate until the initiator is marizes the different polymerization strategies. consumed or deactivated from its source. The main advan- tages are that there is no necessity to modify the template and monomer(s) complex, the obtained particles will have a Bulk or monolith higher surface-to-volume ratio compared with bulk polymer- ization and the template removal will be easier (consequently, Bulk imprinted polymers are obtained with a quick synthetic not requiring strong treatments). However, its main drawback approach that yields a monolith. This monolith material is is that the diluted media leads to heterogeneous cavities based usually grinded and meshed to obtain polymer fractions with in several low-affinity interactions that might affect its selec- a controllable particle size. Due to its simplicity, robustness tivity as MIP. In this manner, Rebelo et al. combined compu- and the possibility to work in a wide range of solvents, pres- tationally studies with precipitation polymerization to develop sure and temperature, this approach is widely used in chro- a voltammetric sensor for furazolidone antibiotic [46]. matographic applications, e.g. for use as column’s stationary Similarly, Gholivand et al. prepared a MIP towards phases in high-performance liquid chromatography (HPLC) methocarbamol, which was later used as selective adsorbent or in solid-phase extraction cartridges (SPE). The main draw- to develop a molecularly imprinted solid-phase extraction, back derives from the monolith treatment. On the one side, either with voltammetric or chromatographic detection [47]. those steps are time consuming and there is a loss of polymer Using the same approach, another voltammetric MIP-based during the grinding and sieving until obtaining a controlled sensor towards histamine was prepared to detect and quantify particle size. On the other side, given the possible damage to histamine in wine samples (Fig. 3) [48]. Another interesting Table 1 Summary of the main advantages and disadvantages of the more common polymerization methods Polymerization Advantages Disadvantages Example of reported method (bio)sensing applications Bulk Simple and cost-effective Harsher template removal and heterogeneity in [44, 45] particle size distribution and/or cavities Precipitation Quick synthesis and easier template removal Diluted reagents media producing heterogeneous [46–49] with high yields cavities Emulsion Small nanoMIPs Emulsifier agent needed, adding an extra synthesis [50–52] step Core-shell Dual property material with outer imprinted Inefficient imprinting if outer layer is too thin [53–55] layer (silica, metallic or magnetic) Herrera-Chacón A. et al. Fig. 3 Confocal microscopy assay to confirm the imprinting effect on the 340 nm. (c) Background fluorescence of the MIP particles when not MIP in respect to the NIP. Images of (a) MIP and (b) NIP particles after incubated with histamine, but reacted with OPA, as negative control. incubation with histamine and reaction with o-phthaldialdehyde (OPA), (d) Fluorescence intensity of the previous in arbitrary units. Reproduced which yields a maximum fluorescence at 455 nm upon excitation at with permission from Elsevier [48] work was published for the detection of BPA by incorporating is the extra step required for its synthesis and the extra-cost in polymerized MIPs into screen-printed carbon electrodes [49]. the laboratory due to the apparatus and the conditions re- quired. The use of magnetic particles as core-shell for a later imprinting is a widely used approach as mentioned by Yañez- Emulsion polymerization Sedeño et al. [53], as it offers the possibility to further pre- concentrate the sample and minimize the matrix effects. For The emulsion approach consists on mixing the organic mono- example, a magnetic-MIP was prepared for the isolation and mer(s), the template and the cross-linker solution with an aque- detection of biotin and biotinylated biomolecules by ELISA ous surfactant media, obtaining a micro- or mini-emulsion. In immunoassays [54]. Another interesting approach was the use order to supress the barrier between the two media, the emulsion of Ru(phen)32+ dye molecules embedded off-centre in the sil- has to be strongly stirred or even sonicated. The main drawback ica core and a thin surface-grafted MIP shell intervening as of this approach is the heterogeneity of the imprinting due to the selective enrofloxacin-binding element for photochemical de- use of water and the surfactant, which can affect the stability of tection of enrofloxacin [55]. the template-monomer(s) interactions, and the use of chemical surfactants that will require purifying steps. Consequently, this is not a very attractive technique for MIP synthesis. Recent exam- ples in the literature using emulsion approach include the prepa- ration of MIPs for their usage in solid-phase extraction (SPE) of Immobilization bisphenols from urine samples [50] or florfenicol from milk [51], As abovementioned, the imprinting technique allows or the imprinting of testosterone [52]. obtaining artificial receptors that mimic the biological ones such as enzymes or antibodies. In order to be used for sensing Core-shell polymerization purposes, these materials have to be integrated into the sensor, where a transducer will convert the recognition (or binding) Core-shell polymerization, as the name suggests, implies the event into an analytical signal. For that purpose, there are two polymerization of the shell of the MIP around a core. This main strategies in the incorporation of MIPs: decoupling the core is not necessarily a non-functional core; it may comprise synthesis and immobilization of the artificial receptor (ex situ different materials depending of the desired application, e.g. synthesis) or achieving the direct polymerization onto the magnetic and metallic nanoparticles or support like glass electrode surface (in situ). The former enables a better optimi- beads to offer a control imprinted with grafting or chemical zation of each step separately, taking often advantage of the grafting. The main advantage of core-shell MIP particles in immobilization step to incorporate new micro- or nano- respect to other approaches is that those offer a better and materials to improve the electrochemical response (i.e. to im- tunable control of the thickness of the polymeric film, which prove the sensitivity or the selectivity). Oppositely, the latter in turn allows a better access of the analytes to the cavities and are generally one-step methods, which permit a better a high binding capability of the material. The main drawback Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues adherence of the film to the transducer surface and a better methidathion [57]. Similarly, several works have been pub- control of the film thickness. lished in our laboratory for the incorporation of different MIP In this section, several immobilization strategies used when particles into voltammetric sensors via sol-gel immobilization developing electrochemical sensors will be reviewed. On the and spin coating [48, 58, 59]. one hand, the incorporation of micro- or nano-materials can be Nafion® has been widely used for the preparation of mod- achieved by different methods such as drop-, spin- or spray- ified electrodes due to its convenient properties such as being coating, or through the preparation of composite materials. On non-electroactive, hydrophilic and insoluble in water. For the the other hand, for the in situ generation of MIP sensors, coating of the electrodes, a solution is prepared in alcohol or chemical grafting followed by a polymerization step or water, to which nanomaterials can be suspended, deposited electropolymerization are the more common approaches. onto the electrode surface, by e.g. drop-casting, and finally dried [60]. Following this approach, a hemin-based co-poly- Ex situ MIP sensors merized MIP prepared by precipitation was deposited onto a glassy carbon electrode (GCE) using Nafion® for the amper- As already stated, decoupling the synthesis and the immobili- ometric sensing of 4-aminophenol [61]. Similarly, a zation steps allows to firstly complete the synthesis and char- voltammetric sensor towards tryptophan was prepared by acterization of the MIP particles, and upon selection of the drop-casting of a mixture of the prepared nanoMIP and optimum conditions, proceed with their integration into the multi-walled carbon nanotubes (MWCNTs) [62]. transducer, being also possible to incorporate at the same time Like Nafion®, chitosan can be used as membrane to electro-catalytic materials that enable to improve the electro- incorporate the MIP nano- or micro-particles as well as chemical response. Common approaches are mainly based on different materials. This material is a linear polysaccha- physical entrapment onto the electrode surface with the aid of ride, obtained by deacetylation of chitin that is also com- some membrane or polymeric material such as sol-gel, monly used for the preparation of modified sensors due to Nafion® or polystyrene, or into the composite material that its biocompatibility, being a toxic-free agent and of low- will act as the working electrode by mixing both before its cost. In this direction, Roushani et al. reported the devel- preparation. opment of a voltammetric sensor towards manganese based on the casting onto a GCE of the synthetized MIP Entrapment onto electrode surface along with an ionic liquid and MWCNTs via drop-casting from a chitosan mixture [63]. The incorporation of MIP particles onto the electrode As already stated, there are a large number of polymeric through its entrapment is probably the common ap- materials suitable to carry out the entrapment of the proach, as is reflected in several publications. There synthetized MIP particles. For instance, poly(methyl methac- are several ways by which these could be done, some rylate) (PMMA) is a synthetic acrylic polymer that can also be of the more popular being the use of sol-gel, Nafion®, used as an entrapment agent. For example, a QCM-based chitosan, agarose, glutaraldehyde, between others. The sensor for volatile organic compounds was prepared by spin main advantage of this approach is that it allows the coating of a dispersion containing the MIP particles and simultaneous incorporation of different materials in a PMMA on an AT quartz crystal resonator [64]. simple and fast way, e.g. MIP beads enhanced with Lastly, electropolymerization of different materials such as graphene oxide sheets and/or other electrocatalysts. poly(3,4-ethylenedioxythiophene) (PEDOT), oPD, pyrrole or The idea is that the particles are retained into polymeric Nafion®, between others, has also been used not only for the frameworks, while maintaining their structure and prop- development of electropolymerized MIPs as discussed in the erties. In order to ensure a homogeneous distribution “In situ electropolymerized MIP sensors” section, but also to along the sensing platform, several techniques can be achieve the immobilization of the MIP particles previously used ranging from drop-casting to spin coating (Fig. 4.). synthetized. The aim is to combine the advantages of Sol-gel is a method that allows the incorporation of differ- decoupling MIP synthesis and immobilisation steps, with the ent materials into a generated inorganic framework. The pro- ones from electrochemical preparation [65]. This approach cess begins with a colloid liquid suspension of an alkoxide was first reported by Ho et al., who used PEDOT to immobi- precursor (“sol”), which is gradually transformed into a “gel” lize the MIP prepared by precipitation polymerization onto an by its hydrolysis, poly-condensation and drying [56]. The indium tin oxide (ITO) electrode when developing an amper- most common alkoxide precursor is tetraethyl orthosilicate ometric sensor towards morphine [66]. Analogous approach (TEOS) due to the possibility to obtain thin-film porous has been later adapted with electrochemically grown films of layers. For example, this approach has been used by Bakas polypyrrole, either after the drop-casting of the MIP particles et al. for the entrapment of a bulk MIP via drop-casting when [65] or directly from a dispersion containing both the particles, developing an impedimetric sensor for the detection of single-wall carbon nanotubes (SWCNTs) and pyrrole [67]. Herrera-Chacón A. et al. Fig. 4 Representation of the immobilization of the MIP particles either by drop-casting or spin coating Entrapment into the electrode composite examples of such an approach can be found in the literature. For instance, a voltammetric sensor for metronidazole was Another option is the incorporation of the MIP particles di- prepared by mixing the graphite powder, n-eicosane and the rectly into the working electrode by mixing the MIP particles MIP particles [68]. Similarly, an impedimetric sensor for the with the material used to prepare the electrode. This is possible determination of digoxin [69] and a potentiometric sensor for with, e.g. carbon paste electrodes (CPEs) or graphite-epoxy the determination of bisoprolol fumarate [70] have also been composites (GECs). The advantage of such an approach is its reported. Not only the incorporation of MIP particles is pos- simplicity and the strong retention of the particles, but unlike sible with traditional sensors, but the deposition of MIPs into the previous polymeric entrapment options, those might offer electrodes might also be possible by inkjet printing, screen a smaller number of MIP particles available due to the differ- printing, soft lithography, contact printing, 3D printing and ence in porosity between the materials. Nevertheless, many roll-to-roll processing [71]. Fig. 5 Schematic of the steps involved in the surface-initiated polymer- linking agents and template, and the photopolymerization of the mono- ization. Briefly, the iniferter (which acts as the initiator, transfer agent and mers under UV irradiation. Reproduced with permission from Estonian terminator) is electrografted to the sensor surface, which is followed by Academy Publishers [74] the addition of the solution containing the functional monomer(s), cross- Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues In situ MIP sensors Electropolymerization In order to address the integration and compatibility between the As already discussed in the “In situ electropolymerized MIP MIP and the transducer, in situ polymerisation of imprinted thin sensors” section, electropolymerization can be used for the in films was proposed. The aim is to promote the formation of situ synthesis of MIPs. The advantage of such an approach is selective binding sites closer to the sensor surface and/or enhanc- the better adherence of the film to the transducer surface and ing the diffusion kinetics [32]. Furthermore, in situ synthesis control of the film thickness. Earlier, an example using oPD as provides better adherence of the film to the transducer surface monomer has already been presented [31]. Similarly, Özcan de- and better control of the film thickness. Over the different ap- veloped a voltammetric sensor for paracetamol based on a poly- proaches that have been proposed, herein we want to highlight pyrrole MIP film electrochemically synthetized by simply two: grafting and electropolymerization. mixing pyrrole, the template and a supporting electrolyte [75]. With this approach, even MIPs towards bacteria have been synthetized (e.g. S. aureus, Fig. 6) [76]. Another interesting ap- Grafting proach is the one proposed by Ma et al. presenting a covalent bond between the protein (template) and glutaraldehyde by imine The attachment of initiators or polymerizable groups through reaction for determining a prostate-specific antigen (PSA) using a different chemistries has been proposed as a successful ap- GCE coated with graphene nanoplatelets and gold nanoparticles, proach for the preparation of ultra-thin MIPs [72]. The main further modified with a MIP (Fig. 7) [77]. methods by which the grafting may be achieved are by utilizing self-assembled monolayers (SAMs) of, e.g. thiols on gold elec- trodes, silanes or diazonium salts. Next, the polymerization reac- Applications tion is done directly on the electrode surface, by dropping a solution containing the template, monomer(s), cross-linker(s) or The number of applications involving the usage of MIPs as initiator depending on which was the immobilized agent. For recognition element for the development of electrochemical example, Khlifi et al. reported the electrografting of 4- sensors is increasingly growing as these polymeric materials benzoylphenyl groups from the corresponding diazonium salt, gain popularity due to the already discussed advantages de- which were used as the photoinitiator for the synthesis of a rived from their usage. Over the wide range of techniques that MIP for melamine detection, offering a covalent binding of the electroanalytical methods include, electrochemical sensors are MIP [73]. With a similar approach, Kidakova et al. reported for developed based mainly on three types of transduction: the first time, a MIP-based synthetic receptor capable of selec- potentiometry (where a difference in potential is measured), tively binding a clinically relevant protein—the brain-derived voltammetry (where a current is measured upon application of neurotrophic factor (BDNF, Fig. 5) [74]. a polarization potential) and electrochemical impedance Fig. 6 Schematic of the sensing approach for S. aureus sensing based on an electropolymerized MIP for 3-thiopheneacetic acid and its determination by EIS measurements of the blockage upon rebinding with [Fe(CN)6]3/4 − as the redox probe. Reproduced with permission from Elsevier [76] Herrera-Chacón A. et al. Fig. 7 Schematic of the fabrication and detection principle of the voltammetric MIP-based sensor towards PSA. Briefly, the GCE was modified with a graphene nanosheet-gold nano- particles (GS-Au) solution, followed by the deposition of chitosan (CS). Next, it was acti- vated with glutaraldehyde (GA) to achieve the covalent immobili- zation of PSA through an imine bond between the two. Lastly, dopamine was electropolymerized to obtain the in situ MIP after removal of PSA by immersion in an acetic acid solution. Reproduced with per- mission from Springer [77] spectroscopy (EIS, where the impedance is measured upon advantages derived from the combination of MIPs with elec- application of different frequency AC potentials). Although trochemical sensors. In this direction, Florea et al. reported the each of the techniques presents their inherent advantages and successful development and application of an electrochemical disadvantages [78, 79], ultimately the suitability of the chosen sensor based on an electropolymerized MIP towards the de- technique will depend on the nature of the target analyte and how termination of 17β-estradiol in river waters [80]. Since the suitable it is for that technique (e.g. if the compound is target compound is not electroactive, the binding with the electroactive or not) and the sensing strategy (direct detection MIP was monitored making use of [Fe(CN)6]3/4− as the redox of the analyte or indirect measurements by measuring the cavities probe. blockage by means of a redox probe such as [Fe(CN)6]3/4−). Analogously, a MIP-based sensor towards cefalexin (an In this direction, herein we want to present some relevant antibiotic) was also developed, and its application to river applications recently reported in the literature over different water and pharmaceutical samples successfully demonstrated scenarios in order to illustrate the potential of MIPs in combi- [81]. Interestingly, in this case, the authors compared the per- nation with electrochemical sensors (Table 2). However, we formance of the same MIP when using a GCE or a boron- want to emphasize that although variants using different tech- doped diamond electrode (BDDE), showing much better per- niques and protocols have been included, the aim is not to formance with the latter, which demonstrates not only the exhaustively review all the applications, but to demonstrate importance of the MIP, but also of the transducer and of the its wide applicability over different scenarios. usage of electrocatalysts when developing electrochemical (bio)sensors. Selectivity of the MIP was evaluated over 11 different cephalosporins, showing only significant interfer- Environmental ence from three of them due to the structural similarities. In the same direction, Rebelo et al. used quantum mechan- As already introduced, the number of emerging contaminants ical calculations and molecular dynamics simulations to opti- is growing continuously, and MIPs represent a very interest- mize the synthesis of a MIP for furazolidone. Polymers were ing approach to allow their on-field detection due to the Table 2 Survey of diverse applications in different fields employing different types of MIP-based electrochemical sensors Analyte Sample1 Sample1 Polymerization Immobilization Electrochemical Electrode Linear range LOD4 Ref.5 method technique2 type3 Environmental 4-Aminophenol Water Spike Precipitation Nafion Amp GCE 10.0 to 90.0 μM 3 μM [61] Bisphenol A (BPA) Buffer – Precipitation Entrapment into ink/composite CV GSPE 0 to 410 ng·L−1 13 ng·L−1 [49] Bisphenol A (BPA) Plastic samples Real Precipitation PVC membrane Pot Graphite 0.5 to 13 μM 0.15 μM [83] paper Cefalexin Water Spike In situ – DPV GCE/BDDE 10 to 100 nM 4.9/3.2 nM [81] electropolymerization Cu(II) Water Spike Suspension SAM of cysteamine and DPV AuSPE 1.9 to 61 nM 74 pM [82] carbodiimide cross-linker chemistry 17β-Estradiol Water Spike In situ – LSV Au 3.6 fM to 3.6 nM 1.09 fM [80] electropolymerization Furazolidone Water Spike Precipitation Entrapment into composite DPV CPE 0.01 to 1 μM 0.03 μM [46] electrode Mn(II) Water Spike Precipitation Chitosan SWASV GCE 2.0 to 9.0 μM 0.15 μM [63] Methidathion Water Spike Bulk Sol-gel EIS GSPE 40 to 200 μg·L−1 5.14 μg·L−1 [57] N-Nitrosodimethylamine Water Spike Bulk Electropolymerization of pyrrole EIS GCE 10 to 230 μg·L−1 0.85 μg·L−1 [67] (NDMA) Food control 4-Ethylphenol Buffer – Precipitation Sol-Gel DPV GEC 5 to 35 μg·L−1 1.33 μg·L−1 [59] 4-Ethylguaiacol Buffer – Precipitation Sol-Gel DPV GEC 3 to 21 μg·L−1 1.55 μg·L−1 [59] Histamine Fish Spike Magnetic core-shell Magneto-capture CV GEC 0 to 2.2·104 mg·L−1 1.6·10−6 mg·L−1 [86] Histamine Wine Real Precipitation Sol-gel DPV GEC 0.5 to 6.0 μg·L−1 0.19 μg·L−1 [48] Lactose Milk Real In situ – DPV Graphite 1.0 to 10 nM 0.88 nM [84] electropolymerization paper Melamine Milk Spike In situ – SWV Au 10−9 to 10−7 M 7.71·10−10 M [73] photopolymerization −1 −1 −1 Prostate-specific Serum Real In situ – DPV GCE 1 pg·mL to 100 ng·mL 0.15 pg·mL [77] antigen (PSA) electropolymerization −6 −4 −7 Sulfamethazine Milk Spike Bulk None SWV GCE 2.0·10 to 4.0·10 M 3.0·10 M [85] Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues Theophylline Buffer – Precipitation Sol-gel DPV GEC – 1 μM [58] Tryptophan Milk, serum and pharmaceutical Spike/real Precipitation Nafion DPV GCE 6.0·10−8 to 4.0·10−5 M 7.1 nM [62] S. aureus Milk Spike In situ – EIS Au 0 to 108 CFU·ml−1 2 CFU·mL−1 [76] electropolymerization Clinical diagnostics Artemisinin Human serum and plant extracts NA In situ – CV/SWV Au 0.01 to 1.36 μM 0.01 μM [90] electropolymerization Brain-derived Buffer – In situ – DPV AuSPE 0.01 to 0.06 ng·mL−1 6 pg·mL−1 [74] neurotrophic factor photopolymerization (BDNF) Bisoprolol fumarate Pharmaceutical, urine and blood Real Bulk Entrapment into composite Pot CPE 1.0·10−7 to 1.0·10−2 M 5.0·10−8 M [70] electrode C4-HSL Buffer – Bulk/SPE SAM of cysteamine and DPV AuSPE 6.25 to 800 nM 0.12 nM [71] carbodiimide cross-linker chemistry Daptomycin Human serum Spiked In situ – DPV GCE 1 to 20 pM 0.161 pM [91] electropolymerization −9 −7 −11 Digoxin Human blood serum Spike Bulk Entrapment into composite EIS CPE 1.0·10 to 0.5 10 M 6.95·10 M [69] and pharmaceutical electrode Dopamine/ascorbic acid Serum NA FRP DPC GSPE 100 to 500 nM – [93] Doxycycline Pharmaceutical preparations Real Bulk PVC membrane Pot Perspex 7.99·10−6 M to - 3.55 μg·mL−1 [87] Ephedrine Buffer – Precipitation CV GCE 0.5 to 3 mM – [65] Ferritin Urine Spike and In situ – CV Au 40 to 360 mg·dm−3 10.7 mg·dm−3 [89] Real electropolymerization Glucose Buffer – Bulk/SPE SAM of cysteamine and DPV AuSPE 0.8 to 50 mM 0.43 mM [71] carbodiimide cross-linker chemistry Table 2 (continued) Analyte Sample1 Sample1 Polymerization Immobilization Electrochemical Electrode Linear range LOD4 Ref.5 method technique2 type3 Human serum Urine Spike and In situ – CV Au 20 to 100 mg·dm−3 3.7 mg·dm−3 [89] albumin (HSA) real electropolymerization Levothyroxine hormone Synthetic samples Spike Magnetic core-shell Direct magneto immobilization LSV GEC 3.88·10−6 to 0.0356 ng·mL−1 [92] 388.4 μg·mL−1 Methocarbamol Human plasma Spike Precipitation None DPV GCE 0.10 to 15.0 μg·mL−1 0.025 μg·mL−1 [47] Metronidazole Human serum and urine Spike Bulk Entrapment into DPV CPE 5.64·10−5 to 3.59·10−5 mg·L−1 [68] composite electrode 2.63·10−3 mg·L−1 Naloxone Urine/serum Spike In situ – DPV GSPE 0.25 to 10 μM 0.20 μM [88] electropolymerization −7 Paracetamol Pharmaceutical Spike In situ – DPV PGE 5 μM to 0.50 mM 7.9·10 M [75] electropolymerization Paracetamol Buffer – Bulk/SPE SAM of cysteamine and DPV AuSPE 100 to 1000 μM 82 μM [71] carbodiimide cross-linker chemistry SARS-CoV-2 antigen Lysis buffer Spike and In situ – DPV Au 2.22 to 111 fM 15 fM [94] real electropolymerization −14 −9 −14 Trypsin Buffer – Solid-phase method Electropolymerization of Cap Au 1·10 to 1·10 M 1·10 M [95] tyramine, followed by glutaraldehyde Trypsin Buffer – Bulk/SPE SAM of cysteamine and DPV AuSPE 6.5 to 100 nM 0.20 nM [71] carbodiimide cross-linker chemistry Homeland security and forensic Cocaine Saliva, river water and street Spike In situ – SWV GphSPE 100 to 500 μM 50 μM [96] samples electropolymerization 3,4-Methylenedioxy Plasma and urine Spike In situ – SWV GSPE 2.6 to 200 μM 0.79 μM [97] methamphetamine electropolymerization (MDMA) Morphine Buffer – Precipitation Electropolymerization Amp ITO 0.1 to 2 mM 0.3 mM [66] of PEDOT −12 −5 −14 Tetrahydrocannabinol Buffer – Solid-phase method Electropolymerization of tyramine, Cap Au 1·10 to 1·10 M 1·10 M [95] (THC) followed by glutaraldehyde Tetrahydrocannabinol Buffer – Bulk/SPE SAM of cysteamine and DPV AuSPE 0.1 to 1000 μM 0.05 μM [71] (THC) carbodiimide cross-linker chem- istry 2,4,6-Trinitrotoluene (TNT) Buffer – In situ – CV Au 0.1 to 10 nM 50 pM/100 pM [41] electropolymerization RDX Buffer – In situ – CV Au 0.1 to 10 nM 100 pM [41] (1,3,5-trinitroperhydro-1, electropolymerization 3,5-triazine) 1 “Buffer” refers to sensors that have only been tested in buffered media with stock solutions, “spiked” to sensors that have been tested in spiked real samples, “real” to sensors which performance has been satisfactorily validated in real samples, and “NA” to sensors that were tested in real samples, but its performance not benchmarked 2 Amp, amperometry at fix potential; Cap, capacitive via current pulse method; CV, cyclic voltammetry; DPC, double-pulse chronoamperometry; DPV, differential pulse voltammetry; EIS, electrochemical impedance spectroscopy; LSV, linear sweep voltammetry; Pot, potentiometric; SWV, square wave voltammetry; SWASV, square wave anodic stripping voltammetric 3 AuSPE, gold screen-printed electrode; BDDE, boron-doped diamond electrode; CPE, carbon paste electrode; GCE, glassy carbon electrode; GEC, graphite-composite electrode; GphSPE, graphene screen-printed electrode; GSPE, graphite screen-printed electrode; ITO, indium tin oxide; PGE, pencil graphite electrode 4 LOD, limit of detection 5 Ref, reference Herrera-Chacón A. et al. Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues mixed with graphite and MWCNTs to obtain the CPE, which manner a significant pre-concentration of the analyte (authors was used to measure the content of furazolidone in tap and reported an enrichment factor of 45), while also removing river water samples without spiking or doing any pre- possible interferents. In their study, recoveries of ca. 100% treatment [46]. were achieved for the different spiked milk samples. In a similar approach, a MIP-based impedimetric sensor Another approach to deal with complex samples is the was developed for the detection of N-nitrosodimethylamine combination of MIPs with magnetic particles, which allow (NDMA) in tap water [67], choosing impedimetric measure- faster pre-concentration while also minimizing matrix effects. ments over voltammetric given its higher sensitivity. The MIP For example, Hasan et al. presented the development of a particles were first synthetized by the precipitation polymeri- core-shell magnetic-MIP for the electrochemical sensing of zation method, and next entrapped in an electropolymerized histamine in fish samples (Fig. 9) [86]. The developed MIP polypyrrole layer. The imprinting effect was confirmed from demonstrated high selectivity as almost no interference was the sensitivity of the MIP which was 8.3-fold higher than that observed for the other three more common biogenic amines. of the NIP, as well as from the much smaller response (vari- Moreover, recovery values when the sensor was applied to the ation of charge transfer resistance) obtained towards structural analysis of fish samples ranged from 96.8 to 102.0%, analogous compounds. confirming that this approach can be used for routine food As already stated, MIPs can be even developed towards examination. small compounds as metallic ions. For example, Di Masi Recently, da Silva et al. reported the electropolymerization et al. reported the synthesis and characterisation of ion- of a MIP on a disposable graphite paper electrode [84]. imprinted polymer nanoparticles with embedded ligand sites Concretely, towards the determination of lactose in milk sam- and their application for the electrochemical determination of ples (Fig. 10). The MIP showed good imprinting in compar- Cu(II) [82]. Synthetized MIPs were immobilized on gold ison to the NIP, good selectivity towards structural analogues screen-printed electrodes (AuSPE) by carbodiimide chemistry as well as good repeatability and reproducibility. Even lactose after formation of a SAM with cysteamine. The developed is non-electroactive; detection was accomplished through MIP showed good response towards Cu(II), with a sensitivity blocking of conducting channels across the MIP layer and ca. 7.5 times higher than the NIP (confirming the imprinting), oxidation of a ferrocyanide marker observed by DPV. but selectivity coefficients of 0.66–0.68 for Ni(II) and Zn(II), However, poorer stability was observed for measurements which was attributed to the three of them having a similar across several days, which is not as bad if considering the ionic radius. In this direction, it has to be highlighted that disposable nature of the paper sensor. despite MIPs can be developed towards almost any com- pound, it is true that the imprinting of smaller compounds is Clinical diagnostics hindered by the more limited interaction sites that those offer. MIPs have also been used along with potentiometric sen- In another example involving potentiometric sensors, Kamel sors by mixing the synthetized particles into the ion-selective et al. reported the successful determination of doxycycline in electrode (ISE) membranes. For example, Kamel et al. report- pharmaceutical tablets using a MIP-based potentiometric sen- ed for the first time a simple and ultra-low-cost, disposable sor integrated into a flow injection analysis (FIA) system [87]. paper-based potentiometric sensor for determination of neutral The sensor was chosen between three different MIPs prepared BPA [83]. Paper substrates are accessible, flexible, recyclable with different monomers, all of them showing good response, and biodegradable, making them an attractive material for the whereas the respective NIP sensors’ did not exhibit a linear preparation of electrochemical MIP sensors [83, 84]. The sen- response in the considered range. sors were built employing commercial chromatography paper, An interesting study presented the development of a MIP- into which the synthetized BPA-MIP nanobeads were mixed based sensor to be used as a point-of-care device for people with the PVC membrane cocktail and incorporated by drop- with opioid overdose from urine or serum analysis [88]. To casting (Fig. 8). Finally, the proposed sensing platform was this aim, a MIP against naloxone was generated by in situ successfully applied to determine BPA released from real electropolymerization of 4-aminobenzoic acid (4-ABA) on plastic samples. top of a commercial graphite screen-printed electrode (GSPE) previously modified with MWCNTs. Despite some Food control interference was observed for structural analogous com- pounds, satisfactory recoveries were obtained for spiked urine Now in the field of food control, an alternative application was and serum samples. Other examples of MIP-based sensors to described by Guzmán-Vázquez de Prada et al., in which MIPs be used as point-of-care devices have been reported by were employed as selective solid-phase extraction sorbent and Stojanovic et al. for the determination of human serum albu- coupled with separate voltammetric detection for the determi- min (HSA) and ferritin in urine samples of patients with albu- nation of sulfamethazine in milk [85]. Achieving in this minuria [89], by Waffo et al. for the determination of Herrera-Chacón A. et al. Fig. 8 Schematic representation of the main steps involved in the Finally, the membrane cocktail containing the MIP particles was drop- development of a MIP-based potentiometric sensor for the determination casted onto the build sensor surface. Reproduced with permission from of bisphenol A (BPA). Briefly, the process starts coating chromatography Royal Society of Chemistry [83] paper with a carbon ink, followed by its coating with an adhesive plastic. artemisinin in human serum samples [90], or by Ozcelikay method providing a thoughtful strategy for replacing et al. for the determination of daptomycin also in human se- antibodies. rum samples [91]. A particular application was described by Rapini et al. A complete study for the selective recognition of where a nanoMIP was used as sequestering (masking) agent, levothyroxine hormone with a magneto-actuated immunoas- to suppress the signal coming from interfering molecules and say and electrochemical sensing was presented by Moura et al. facilitate the detection of the target analyte [93]. Concretely, [92]. Firstly, theoretical studies were carried out by DFT to suppression of ascorbic acid interference, when attempting the choose the best monomers, and among those, the performance determination of dopamine in serum samples, was achieved of the best three candidates was experimentally compared. by adding the nanoMIP into the electrochemical cell when the Finally, the selected MIP was used to develop the electro- concentration of the former was under 50 nM. Besides the chemical sensor which was compared with the gold standard actual results, what is especially significant is the novelty of Fig. 9 Schematic illustration of the pre-concentration and electrochemical sensing of histamine based on the usage of a magnetic-MIP and a magnetic- GEC electrode. Reproduced with permission from Elsevier [86] Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues Fig. 10 Schematic representation of the main steps involved in the cavities complementary to the template. Sensing is based on the measure- development of a MIP-based voltammetric sensor for the determination ment of pore blockage with [FE(CN)6]3− upon rebinding of lactose when of lactose. Briefly, the process starts with the electropolymerization of the sensor is incubated with the samples. Reproduced with permission pyrrole in the presence of lactose. Next, the template is removed by from Elsevier [84] overoxidation in a NaOH solution, leaving three-dimensional imprinted the application itself, which can be extrapolated to other as good as that for the protein, as could be expected due to the analytes. difference in size between both molecules and, consequently, Lastly, in a very hot topic nowadays with the current the number of functional groups to generate the imprint. COVID-19 pandemic caused by SARS-CoV-2 coronavirus, Many other examples can be found for the detection of Raziq et al. reported the development of an accurate and cheap illicit drugs. A remarkable study was published by Florea portable sensor based on a MIP-based voltammetric sensor et al. [96] for the sensing of cocaine in real samples. The and the usage of a portable compact potentiostat which can MIP was based on the in situ electropolymerization of p- be controlled through a smartphone (Fig. 11) [94]; all of them aminobenzoic acid (which was selected by computational crucially important characteristics for the clinical diagnosis. modelling) onto graphene-modified electrodes with palladium The MIP sensor was able to signal the presence of SARS- nanoparticles integrated into the sensing layer to enhance the CoV-2 nucleoprotein (ncovNP) in nasopharyngeal swab sam- number of accessible imprinted sites, to improve their homo- ples of COVID-19-positive patients, unlocking in this manner geneous distribution and to provide a catalytic effect. The new routes for the development of rapid COVID-19 diagnos- selectivity of the developed MIP was demonstrated against tic tools. homatropine (an alkaloid with similar structure) showing no response, and recoveries of ca. 100% in spiked river water and Homeland security and forensic saliva. Similarly, the detection of 3,4- Canfarotta et al. reported a manufacturing-friendly protocol methylenedioxymethamphetamine (MDMA) has been achieved for the synthesis of MIP nanoparticles produced by solid- by using oPD as the monomer to be electropolymerized onto phase synthesis and their integration into label-free capacitive GSPE, chosen also after DFT calculations to optimize the poly- sensors [95]. The latter chosen due to the higher sensitivity mer structure as well as the interactions of the monomer and the that those offer compared with other techniques, as well as template [97]. The MIP-based sensor showed good selectivity, offering label-free measurements. To prove the feasibility of showing only dopamine and tyramine some binding with the the approach, two different target analytes with different mo- MIP, but being eletroanalytically distinguishable. This was con- lecular weight were evaluated: tetrahydrocannabinol (THC) firmed from the analysis of spiked urine and plasma samples, and trypsin (a protein). Significant performance was obtained where recoveries of 81–91% were obtained. for trypsin, with limits of detections (LODs) down to 10−14 The detection of explosive compounds is another hot topic and high selectivity compared to the response obtained for in forensic analysis due to the surge of international terrorism other proteins, while the performance of THC-MIP was not and the increased use of explosives in terrorist attacks. In Herrera-Chacón A. et al. Fig. 11 Schematic of the steps of the COVID-19 diagnostics principle by based on pore blockage of the [Fe(CN)6]4−. Reproduced with permission ncovNP sensor analysing the samples prepared from nasopharyngeal from Elsevier [94] swab specimens of patients, and of the electrochemical measurement addition, the inherent redox activity of nitro-aromatic com- As a result of the interest that MIPs have arisen, significant pounds makes them ideal candidates for electrochemical progress has been made over the last two decades in the de- methods thanks to their multi-peak reduction signal [98]. In velopment of new synthetic and polymerization approaches this regard, Leibl et al. described the electropolymerization of that enhance the performance of the obtained polymers. In this two dummy MIPs for 2,4,6-trinitrotoluene (TNT) and RDX direction, it must be highlighted the emergence of (1,3,5-trinitroperhydro-1,3,5-triazine) using trimesic acid and electropolymerization which allows a facile approach for the Kemp’s triacid as structural analogues [41]. Moreover, the obtaining of electrochemical-based MIPs, the role of compu- choice of dopamine as the functional monomer was based tational approaches which allow a rational design of MIPs, on DFT computational design using the binding energies for reducing significantly the experimental screening of the best complex formation with both the dummy and the actual target candidate, or the appearance of alternative synthetic ap- as ranking parameter. The developed MIP-based sensor proaches such as dummy MIPs, which provide an experimen- allowed for a 105-fold sensitivity improvement over a bare tal alternative to obtain MIPs towards specific analytes that gold electrode, demonstrating the advantage derived from otherwise would be cumbersome. Furthermore, the combina- the usage of MIPs. tion of nanomaterials with MIPs and sensors allows enhancing the properties of the synthetized polymers as well as the elec- trochemical response of the latter, giving rise to hybrid mate- rials with new properties and higher potential. For example, Conclusions and future trends recently, Piletsky et al. reported on a nanoMIP that incorpo- rates ferrocene as the redox probe, which upon binding of the The combination of MIPs with electrochemical sensors for the template generates a conformational change that can be de- development of new sensing devices has been explored in this tected electrochemically even if the target analyte is non- review. More specifically, an overview of the more common electroactive (Fig. 12) [71, 99]. Precisely, the latter is a good synthetic approaches as well as the strategies that can be used example of the efforts made to produce protocols that allow to achieve their integration has been presented, followed by this technology to reach the market, which as discussed in some recent examples over different areas in order to illustrate more detail by Lowdon et al., is being hindered by a combi- the potential of such combination in very diverse applications. nation of factors such as the challenge that represents the Overall, demonstrating the advantages that can be derived scale-up production process or the fact that lab-based analysis from the usage of MIPs, which show comparable performance using techniques such as liquid or gas chromatography remain to affinity receptors such as enzymes or antibodies in terms of as the gold standard [100]. selectivity, but with the added advantage of its higher stability, However, despite the huge potential that MIPs have dem- lower cost, tunability to almost any template and, more impor- onstrated, it can be neglected that as any sensor or biosensor, tantly, more versatile option in terms of operation conditions those might still show certain cross-response and/or suffer (e.g. extreme temperatures or pH levels and organic solvents). Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues Fig. 12 (Top) Schematic representation of the response principle of the MIP-based sen- sor. Briefly, the rebinding of the analyte to the MIP triggers a de- tectable change in the polymer conformation thanks to the ferro- cene that is incorporated into the polymeric matrix. (Bottom) Scanning electron microscopy images of the synthetized MIPs for paracetamol detection. Reproduced with permission from Springer [71] from undesired matrix effects. To overcome such issues, the biosensors, in what has been referred to as bioelectronic straightforward thought is on trying to improve the tongue (BioET) [102, 103]. synthetized material, but higher benefits can be even obtained An ET is a biomimetic system applied to the analysis of if applying chemometric methods (an approach known as liquid samples, which consists in the coupling of an electronic tongue, ET). array of sensors capable of giving a wide and complete Such an approach was first reported in 1985, when Otto response of the analysed species, plus a chemometric and Thomas attempted the determination of free metal ions processing tool able to interpret and extract meaningful with an array of non-specific ISEs, and demonstrated the ad- data from the complex readings [102, 104]. BioETs on- vantages that could be obtained from the use of partial least ly differ from ETs in the fact that one or several bio- squares regression (PLS) in comparison to ordinary least sensors are combined into the ET sensor array, com- squares regression (OLS) [101]. A few years later, the same monly sharing the same transduction principle to facili- approach was employed to improve the performance of tate their compatibility. Herrera-Chacón A. et al. Fig. 13 Schematic representation of the steps involved in the ET voltammogram for each of the samples. Next, the data is processed by approach reported by Herrera-Chacon et al. [59]. Briefly, the set of sam- means of PCA to achieve the discrimination of the different volatile ples are measured with the MIP-based sensor array, collecting a whole phenols or ANNs for the quantification of volatile phenols mixtures In this direction, the incorporation of MIP-based sensors Sharif et al. showed the potential of using haemoglobyn MIPs into ET sensor arrays would be of great interest, due to the for differentiating meat species [108]. In another study, already discussed advantages of the former, allowing to un- Chatterjee et al. developed a polyacrylamide MIP sensor to- leash new applications and opening the room for the develop- wards theaflavin, and attempted the correlation of the ment of what could be considered a new type of ETs based on voltammetric readings with the theaflavin content estimated its usage. On the one side, the incorporation of MIP-based by HPLC by means of partial least squares regression (PLS) sensors into ETs allows to address the lack of selectivity of employing a leave-one-out (LOO) approach; the study demon- the latter in a similar way that biosensors do, but offering strated good agreement between both, although further validation lower cost systems with higher stability and operability in might be required due to the reduced number of samples and the harsher conditions, while allowing to obtain a suitable recep- use of LOO validation [109]. Lastly, in our laboratory, the appli- tor towards almost any analyte of interest. On the other side, cation of a MIP-based ET towards the determination of volatile the modelling stage involved in ETs allows to solve possible phenols in wines with the Brett defect was also reported [59]. In interferences and to improve the selectivity of the synthetized this case, the ET array was formed by 3 sensors modified with MIPs, by shifting the complexity from the chemical to the a MIP towards 4-ethylphenol, another towards 4-ethylguaiacol modelling side [102, 104, 105]. However, despite the high and the NIP, achieving the resolution and individual quantifica- number of publications related to the development of MIP- tion of binary mixtures of both volatile phenols by means of based electrochemical sensors, very few publications can be artificial neural networks (ANNs) (Fig. 13). found exploiting this approach [106]. One of those few studies is the one from Bueno et al. where an array of four hydrogel-based MIPs towards different pro- Funding The authors acknowledge the support of the Spanish Ministry of Science and Innovation (MCINN) through project PID2019- teins (bovine haemoglobin, equine myoglobin, cytochrome C 107102RB-C21. A.H.C. thanks the support of the projects CTQ-2016- and bovine serum albumin) was prepared using GCEs. The 80170-P. M.d.V. thanks the support from Generalitat de Catalunya voltammetric responses were submitted to PCA in order to through the program ICREA Academia. attempt their discrimination, showing clear clusters for each of the proteins. However, despite the same trend was observed Data availability Not applicable. for the NIP, it opened the door for future applications [107]. Precisely, in a very similar approach with spiked samples, El- Code availability Not applicable. Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues Declarations 19. Wulff G, Sarhan A. Use of polymers with enzyme-analogous structures for resolution of racemates. Angew Chem Int Ed. 1972;11:341–4. Conflict of interest The authors declare no competing interests. 20. Sagiv J. Organized monolayers by adsorption. III. 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