Molecularly Imprinted Polymer-Based Electrochemical Sensing in Protein Detection

. Protein detection is paramount across various scientific, clinical, and industrial domains. Accurate and sensitive detection of proteins is pivotal for understanding biological processes, diagnosing diseases, drug development, environmental monitoring, and ensuring food safety. Traditional protein detection methods encounter sensitivity, specificity, and ease of use challenges. Molecularly imprinted polymers (MIPs), with their tailored molecular recognition sites, offer a novel approach to address these limitations. When combined with electrochemical techniques, MIP-based electrochemical methods have emerged as a revolutionary technology, showcasing enhanced sensitivity and selectivity. This article provides a comprehensive overview of MIP-based electrochemical methods for protein detection, including the principles, engineering aspects, advantages, and potential applications. The aim is to elucidate the potential of this cutting-edge technology in reshaping protein detection and its promising role in advancing


INTRODUCTION
In the expansive landscape of biotechnology and biosensing, the accurate detection and analysis of proteins stand as a foundational pillar, wielding immense significance across scientific, clinical, and industrial domains.Proteins, as fundamental biomolecules, play a critical role in various biological processes and are integral to disease diagnosis and therapeutic interventions [1,2].Accurate protein detection is essential for deciphering complex biological mechanisms and identifying potential disease biomarkers.Traditional protein detection methodologies, such as enzyme-linked immunosorbent assays (ELISAs) and mass spectrometry, have limitations related to sensitivity, specificity, and complexity of oper-ation [3].MIPs offer a viable alternative with their ability to provide high specificity and binding affinity towards target proteins.When integrated with electrochemical techniques, MIPbased electrochemical sensors provide a potent platform for precise and reliable protein detection [4,5].
Traditional protein detection methods, while invaluable, encounter persistent challenges, ranging from sensitivity limitations to cross-reactivity issues and the need for complex protocols.Addressing these challenges head-on, molecularly imprinted polymers (MIPs) have emerged as a revolutionary technology in analytical chemistry, offering a new dimension to biosensing [6].
MIPs, synthetic polymers with particular molecular recognition sites, have been intricately designed to selectively bind with target proteins, providing a robust foundation for developing sophisticated protein sensors.When coupled with electrochemical techniques, these MIP-based electrochemical methods usher in a new era of protein detection characterised by enhanced sensitivity, selectivity, and ease of use [7].This article will explore protein detection, focusing on the innovative utilisation of MIP-based electrochemical methods.We will delve into the foundational principles and intricate mechanisms that govern MIP-based electrochemical sensing, illuminating the science behind the precise engineering of these polymers to recognise and bind a diverse range of proteins [8,9].Furthermore, we will unravel the multitude of advantages that MIP-based electrochemical sensors bring to the fore, including heightened sensitivity, exceptional selectivity, and the potential for miniaturisation, positioning them as promising candidates for applications in point-of-care diagnostics and field-based monitoring [10][11][12].
Through this comprehensive journey, our objective is to showcase the expansive potential of MIP-based electrochemical methods in revolutionising protein detection.By highlighting their capacity to push the boundaries of biosensing technologies, we envision a future where these innovative techniques significantly impact diverse domains, paving the way for advancements that rely on precise protein analysis [13][14][15].
In doing so, we anticipate a world where health, safety, and quality are underpinned by accurate biomolecular scrutiny.This will empower us to forge ahead into a future of remarkable scientific discoveries and societal well-being.As we delve deeper, we will unfold the intricacies of MIPbased electrochemical sensing, elucidating realworld applications, foreseeing the trajectory of this promising field in the years to come, and discussing the challenges that lie ahead.

MIP-Based Electrochemical Sensors: Principles and Mechanisms
Molecular Imprinting Process.The molecular imprinting process involves arranging molecules within a reaction vessel to create a polymer matrix.The functional monomer is responsible for specificity and reactivity, the cross-linking agent provides stability and structure, and the target protein contributes its form and characteristics.Chemical reactions result in a polymer matrix of intricate patterns and connections [16,17].
The functional monomer conforms to the target protein's shape and chemical properties, creating precise binding sites.The cross-linking agent reinforces the polymer structure [18,19] .The resulting polymer matrix accurately replicates the target protein's structure and behaviour.
Molecular imprinting finds applications in drug delivery, biosensors, and environmental monitoring.It enables precise molecular recognition and promises scientific exploration and technological advancement [20][21][22].
Electrochemical Transduction.Electrochemical transduction is a pivotal link between the molecular recognition capabilities of MIP-based sensors and the ability to interpret these interactions in terms of electrical response.The foundation of this process lies in the precise molecular imprinting technique, where the polymer matrix is strategically designed to contain cavities or imprinted sites that possess a solid binding affinity for the target analyte, often a specific protein [23].
Upon exposure to the target protein, the imprinted sites within the polymer undergo a binding event, creating a unique interaction that alters the electronic properties of the polymer.This interaction can affect the flow of electrons, manifesting as current, voltage, or impedance changes.More specifically, the binding event can either hinder or enhance the movement of charge carriers, thereby modifying the electrical conductivity or resistance of the polymer.These alterations in electrical parameters are directly proportional to the concentration of the target protein in the sample [20,21,[23][24][25].
The electrochemical signal generated by these changes is then meticulously measured and analysed.The distinct signal patterns provide valuable insights into the target protein's presence, concentration, or structural changes.Advanced electrochemical techniques, such as cyclic voltammetry [26,27], electrochemical impedance spectroscopy, or chronoamperometry, characterise and quantify these electrical changes precisely.

Engineering MIPs for Protein Detection
Rational Design and Synthesis.Crafting the properties of a MIP involves a comprehensive and strategic design approach encompassing several vital elements.The choice of monomers, pivotal building blocks of the polymer, is a critical aspect, as it dictates the structure, functional groups, and rigidity of the resulting polymer network.The selection of monomers is influenced by their ability to interact with the template molecules and form complementary binding sites.These binding sites are carefully orchestrated to mimic the template proteins' specific spatial and chemical arrangement, optimising binding affinity and ensuring high selectivity for the target molecules [56,57].
Template proteins play a crucial role in guiding the imprinting process.They act as molecular templates around which the polymer forms, imparting their unique structure and molecular information to the monomers.This molecular memory then guides the creation of complementary binding sites within the polymer, ensuring it can selectively recognise and bind to the target molecules of interest [58].
Incorporating suitable cross-linkers is another essential aspect of tailoring MIP properties.Cross-linkers link the monomers, forming a three-dimensional network that gives the polymer stability and structural integrity.The choice of cross-linkers influences the polymer's porosity, flexibility, and overall mechanical properties.A reasonable selection ensures that the resulting MIP exhibits the desired stability and robustness, making it suitable for various applications [59].
Integrating these components using a rational design approach achieves a Molecularly Imprinted Polymer with finely tuned properties.This tailored MIP exhibits exceptional binding affinity and specificity for the target molecules and enhanced stability, ensuring its effectiveness and reliability in diverse applications such as molecular recognition, drug delivery, environmental remediation, and biosensing [60,61].
Surface Modification and Immobilisation.Achieving the optimal performance of MIPs in protein sensors and biosensing platforms necessitates a thorough understanding of the significance of surface modification and immobilisation techniques.The surface modification involves altering the electrode surface's physical and chemical properties to create a favourable substrate for MIP attachment.This alteration is crucial to ensure a robust interface between the electrode and the MIP, enhancing the stability and durability of the MIP structure [62,63].
Various surface modification techniques can be employed to facilitate the effective immobilisation of MIPs.Physical adsorption involves the adsorption of MIPs onto the electrode surface through van der Waals forces, hydrogen bonding, or electrostatic interactions.Conversely, covalent bonding entails forming strong chemical bonds between the functional groups of the MIP and the modified electrode surface, resulting in a more secure and lasting attachment.Layer-by-layer deposition involves the sequential deposition of alternating layers of oppositely charged materials onto the electrode surface, providing a structured and organised platform for MIP immobilisation [64,65].
Proper immobilisation of MIPs ensures their orientation and accessibility, allowing efficient binding interactions with the target proteins.It prevents random exposure or aggregation of the MIPs, thus maintaining their structural integrity and, consequently, their recognition capabilities.The oriented and stable MIPs on the electrode surface are poised to exhibit a high affinity and specificity toward the target proteins, enhancing the overall binding efficiency and sensitivity of the biosensing platform [66,67].
In summary, meticulous surface modification and immobilisation of MIPs are fundamental steps in harnessing their potential for protein sensing applications.These processes contribute to the stability and structural integrity of MIPs, paving the way for particular and efficient protein recognition -an essential aspect in the design and development of advanced biosensing technologies [68,69].

Advantages of MIP-Based Electrochemical Sensors for Protein Detection
Enhanced Sensitivity and Selectivity.MIP-based electrochemical sensors represent a groundbreaking advancement in sensing technology, showcasing exceptional sensitivity and selectivity.The distinct advantage of MIPs lies in their ability to meticulously engineer specific and high-affinity binding sites during the imprinting process, which involves carefully selecting functional monomers and cross-linkers [70].This strategic selection allows for the creation a molecular framework with precisely tailored recognition sites that mimic the target analyte's molecular structure.These sophisticated imprinted sites possess high specificity and selectivity, ensuring optimal binding affinity for the target molecules, even in complex sample matrices [71].
MIP-based electrochemical sensors' precise and deliberate design involves intricate steps, including forming a template-analyte complex, polymerisation, and subsequent template removal, leaving behind complementary binding sites [72].The resulting MIPs boast an impressive level of molecular recognition, enabling them to selectively capture the target analytes, even amidst many potentially interfering compounds.This targeted selectivity significantly minimises false positives and negatives, enhancing the accuracy and reliability of the sensor's output [73].
MIP-based electrochemical sensors find extensive use in practical applications across various fields.In environmental monitoring, these sensors detect pollutants, toxins, and heavy metals in air, water, and soil samples.Their outstanding selectivity ensures precise measurements, allowing for timely and informed environmental management and preservation decision-making [74].Moreover, these sensors are vital in medical diagnostics, facilitating the early detection of biomarkers associated with various diseases, such as cancer, diabetes, and cardiovascular disorders.The tailored imprinting process enables the sensors to identify subtle changes in analyte concentrations, aiding in early intervention and improved patient outcomes [75].Furthermore, MIP-based electrochemical sensors play a pivotal role in the pharmaceutical industry, assisting in drug development, quality control, and pharmacokinetic studies.The highaffinity binding sites allow for accurate quantification of pharmaceutical compounds in complex matrices, ensuring compliance with regulatory standards and guaranteeing the safety and efficacy of medicinal products [76,77].As technology evolves, ongoing research and advancements in molecular imprinting techniques promise even greater precision and efficiency in designing MIPbased electrochemical sensors, further propelling their application and impact across diverse scientific and industrial domains [78,79].
Potential for Miniaturization and Portability.The advent of these sophisticated sensors, designed with a focus on miniaturisation and precision, heralds a transformative wave in medical diagnostics.Their compact form factor and efficiency empower healthcare professionals to perform tests at the bedside, in rural or underserved areas, and even in resource-constrained environments.This decentralisation of diagnostic capabilities ensures quicker results, enabling immediate intervention and personalised patient care [80][81][82][83].
In a scenario where time is of the essence, such as during a medical emergency or disease outbreak, these sensors prove invaluable.A healthcare worker can swiftly obtain vital protein-level data without needing a specialised laboratory, leading to timely diagnoses and tailored treatment strategies.Furthermore, these sensors' ease of use and rapid results make them indispensable in the arsenal against emerging infectious diseases and other health crises [84].
Beyond healthcare, these sensors offer gamechanging potential in environmental monitoring.They can be deployed in various locations, including remote ecosystems or urban centres, providing real-time monitoring of protein markers relevant to ecological health.This real-time data collection not only aids in detecting environmental pollutants but also supports research and policy decisions to preserve our ecosystems and public health [85,86].
As research continues to refine and expand the capabilities of these sensors, we anticipate a cascade of innovations.These innovations could include enhanced multiplexing capabilities, allowing for the simultaneous detection of a broader range of proteins, thus amplifying the scope and applications of these sensors.Integrating these advanced sensors in diagnostics epitomises a new paradigm in healthcare and scientific advancements, promising a brighter and more efficient future [85][86][87].
Applications and Future Prospects.MIP-based electrochemical sensors have showcased substantial promise across a spectrum of crucial applications, underpinning pivotal areas such as clinical diagnostics, environmental monitoring, and food safety -their potential lies in their ability to detect specific target molecules with high sensitivity and precision selectively.In clinical diagnostics, MIP-based sensors show promise for revolutionising disease detection by enabling multi-target detection, allowing for a comprehensive analysis of biomarkers associated with various diseases.This capability is crucial for early disease identification and monitoring complex conditions, enhancing the effectiveness and efficiency of healthcare systems [88].
In environmental monitoring, MIP-based electrochemical sensors demonstrate their versatility by enabling the detection of pollutants, toxins, and hazardous substances in air, water, and soil.Their high selectivity allows for precise measurements, aiding in enforcing environmental regulations and policies.Moreover, the potential integration of these sensors with remote monitoring systems can provide real-time data, facilitating rapid response and mitigation strategies in environmental emergencies [89].
In the context of food safety, MIP-based electrochemical sensors offer a reliable means to detect contaminants, pollutants, and pathogens in food products.This is essential for maintaining food quality and safety standards ensuring consumer well-being.With the advancements in nanotechnology, these sensors could become even more sensitive and selective, paving the way for better traceability and control throughout the food supply chain [90,91].
Future advancements in MIP-based electrochemical sensors are anticipated to drive their capabilities further.Multi-target detection, where a single sensor can detect multiple analytes simultaneously, is an area of active research and holds immense promise for enhancing sensing efficiency and reducing overall costs.Real-time monitoring capabilities will evolve, enabling continuous data collection and analysis, thereby supporting timely decision-making and intervention [92][93].
Integrating MIP-based electrochemical sensors with emerging technologies such as wearable devices is a frontier with great potential.This integration could lead to wearable sensors capable of real-time monitoring of various biomarkers, providing individuals with personalised health insights and enabling proactive healthcare management.The resulting data could be seamlessly integrated into healthcare systems, empowering individuals and healthcare professionals with a comprehensive view of health status and trends [92,93].
In summary, the trajectory of MIP-based electrochemical sensors is on the cusp of a transformative phase, moving towards a more sophisticated, integrated, and impactful future.These sensors are set to play a pivotal role in revolutionising how we perceive and interact with critical aspects of our lives, health, environment, and overall well-being.

Protein detection through MIP-based electrochemical sensors
Monomer and template mixture.The development of MIP for the electrochemical sensing of proteins has been achieved through several methods, such as in situ bulk polymerisation of monomer-template mixtures andol-gel; precipitation, emulsion and suspension chaining of monomers [94].Although each of these approaches targets specific goals, such as improved electrostatic interactions between templates and the MIP, it is acknowledged that the susceptibility of proteins to denaturation upon sudden shifts of pH and temperature hinders some polymerisation strategies [95,96].Therefore, several authors relied on the bulk electro-polymerisation of monomer-template mixtures to assemble MIPs for protein electroanalysis to keep the conformational integrity of peptide chains [97].
Figure 1 showcases the basic outline of bulk polymerisation of a monomer-template mixture on the surface of an electrode.B -followed by the removal of the template, thereby yielding the cavities C -the protein structure is lysozyme, stored under the code 4LZM at the Protein Data Bank.
The versatility of bulk electro-polymerisation allows this method to be used in the facile modification of working electrodes.Several electrode platforms based on carbon and gold were proven modifiable through this technology.For instance, in a particularly recent outreach, a screenprinted carbon electrode was quickly and reliably modified through bulk electropolymerisation with pyrrole as functional monomer and interleukin-6 as template, thence yielding a sensor with a meagre limit of detection of 0.02 pg ml -1 [98].Similarly, other authors used a screen-printed gold electrode which had its surface modified by bulk electro-polymerisation with phenol and human epidermal growth factor to develop an MIP-based electrochemical sensor for breast cancer, which yielded a detection limit that rivalled that of standard techniques, being the sensibility of the sensor of 1.6 ng l -1 [99].
Nonetheless, the success of bulk electropolymerisation in protein electroanalysis is attributed to the possibility of crafting and tuning the sensitivity and selectivity of sensors by optimising the electrostatic interactions between the MIP and the template.In this regard, it has been reported that the biomimetic properties of MIP allow interaction sites that mimic the behaviour of natural ligand-macromolecule systems, thereby involving Van der Walls and π-π stacking interactions [100].These hydrophobic sites seal off water molecules in the interaction site and lead to a solventless environment of low dielectric constant, which promotes a strong bond effect [100], such as showcased in a recently published MIP-based sensor for prostate-specific antigen that achieved an optimal equilibrium dissociation constant of about 1.02 ± 0.54 10 -14 mol l -1 .This sensor yielded a detection limit that surpassed the golden standard methods for this cancer biomarker, reaching as low as 3.0 10 -8 ng ml -1 [101].At the same time, the exploitation of electrostatic interactions between MIP-template was used to craft highly sensible sensors for cancerrelated inflammatory biomarkers such as interleukin-8.In a particularly recent outreach, this biomarker was determined through a poly (3,4ethylenedioxythiophene) polystyrene sulfonate/ 4-aminothiophenol/ eriochrome black T ensemble, which led to a limit of detection of 1.5 pmol l - 1 [102].
Notwithstanding, bulk electro-polymerisation is known to have limitations regarding the imprinting of more significant and less flexible proteins due to the difficulty in controlling the orientation of the template during MIP formation, which, therefore, can also impair selectivity and sensibility for sensing applications [103].It is known that binding sites for protein recognition occur in defined contact points which cover surfaces between 500 and 3500 Å 2 [100].In this regard, more homogenous binding sites in the MIP increase the contact area, decreasing the free energy change by a measurement unit, culminating in stronger interactions and higher stability [100].As such, alternative methods like surface imprinting have been used to improve the selectivity and sensibility of MIP-sensors for protein detection to address the need to establish more controlled imprinting.
Overall, bulk polymerisation is advantageous because it usually requiresess strenuous protocols to develop electroanalytical sensors for protein detection whilst yielding adequate performance for a wide range of templates [104].Nevertheless, alternatives to bulk electro-polymerisation, such as surface imprinting technologies, are becoming more common due to the need for more control of the imprinting process and more selective and sensible platforms for protein electroanalysis.
Surface imprinting of proteins.The surface imprinting approach for MIP synthesis relies on embedding the template on the working electrode surface.This process can be achieved by anchoring agents that showcase terminal moieties with affinity to the electrode surface and the template [105].The functionalisation of electrode surfaces with anchoring agents before MIP assembly is frequently achieved through chemoadsorption, with self-assembled monolayer (SAM) formation being the most common approach [106].
The anchoring of the template on the electrode surface allows better control of the imprinting process, as the template orientation can be optimised according to the anchoring protocol used to develop the sensor.In fact, as aforementioned, several researchers have proven that surface imprinting leads to more homogenous binding sites [107,108].For instance, a recent report compared the analytical performance of sensors built through bulk electro-polymerisation or surface imprinting of lysozyme with scopoletin as a monomer.The results evidenced that the surface functionalisation with the template before MIP assembly led to an increase in the imprinting factor of almost 3-fold, thereby leading to a more reliable quantification [109].
The most common material for surface imprinting in MIP-sensor development is gold due to the possibility of spontaneous SAM formation, which leads to simpler surface modification protocols and more straightforward manufacturing of highly sensible sensing platforms.For instance, a recent work reported the facile anchoring of SARS-CoV-2 nucleoprotein onto a thin-film-based gold electrode through a self-assembled 4-amino thiophenol monolayer, thereby yielding a meagre limit of detection of 15 fmol l -1 [110].
On the other hand, although not providing spontaneous monolayer formation, carbon materials have also been used in the surface imprinting of proteins by exploiting π-π stacking interactions between anchoring agents bearing aromatic moieties or by forcing the genesis of polar anchoring sites through oxidative treatments such as electrochemical activation [111].For instance, the surface imprinting of troponin T was reported on electrodeposited polyethene blue at multiwalled carbon nanotubes, whilst Dengue virus non-structural protein was surface-imprinted on screen-printed carbon electrodes coated with electrospun nanofibers of polysulfone to develop MIP-based electrochemical sensors for biomedical applications which yielded limits of detection of 0.04 pg ml -1 for troponin T and 0.30 ng ml -1 for Dengue virus protein, respectively [112,113].
Regarding the surface imprinting of proteins on gold electrodes, it is well reported that anchoring agents bearing hydroxyl, thiol, and amino motifs allow the spontaneous formation of monolayers of conserved geometric orientation that favour biosensing design.For instance, a recent outreach described the quick and easy functionalisation of gold electrodes with prostate-specific antigen, followed by the polymerisation of dopamine for MIP formation.The resulting electrochemical sensor was very sensible, with a detection limitf 1.0 pg ml -1 , and showcased a highly ordered topology, which contributed to an opti-mal selectivity towards the antigenic analyte [114].
The affinity of selected functional groups to gold surfaces allowed researchers to immobilise proteins onto outer layers of electrode materials, promote the in vitro carbamylation of amino acid residues, and provide antifouling interfaces for MIP-based electrochemical biosensors [115].Nonetheless, the versatile assembly of SAMs on gold electrodes for surface imprinting has also given rise to multi-analyte point-of-care biosensors for healthcare applications, such as the concomitant use of prostate-specific antigen and myoglobin as surface imprinted templates for poly-acrylamide MIP assembly in a dual-sensing impedimetric biosensor; leading to detection limits of 5.40 pg ml -1 and 0.83 ng ml −1 , respectively [116].
Concerning the length of SAM-forming anchoring agents, it has been shown that short-chained SAM-forming units allowed the development of highly sensible biosensors for the detection of peptides such as oxytocin, whose anchoring on a gold electrode surface was mediated by allyl mercaptan, followed by MIP synthesis through 2hydroxyethyl methacrylate-methacryloyl amidoglutamic acid; which yielded the sensor with a limit of detection of 0.0030 ng ml -1 [117].Nevertheless, the use of lengthier-chained thiol-bearing residues has also been reported to produce reliable sensors, as evidenced by the use of 11mercaptoundecanoic in many sensing platforms, as well as the immobilisation of double-cysteinemodified peptide nanofilms onto gold surfaces for neuron-specific enolase imprinting on polyscopoletin-based MIP, which yielded a detection limit of 0.25 μmol l -1 [118].
Overall, surface imprinting technology in protein electroanalysis is conditioned by the intrinsic features of the electrode surface and those of the anchoring agent and the template.Therefore, the appropriate protocol must consider the surface material, the chemical structure and topology of the anchoring agent, and their compatibility with the template.Nevertheless, overly bulky templates may hinder the application of surface imprinting for whole-protein sensing, which led to the development of alternative imprinting approaches such as epitope imprinting.
Epitope Imprinting.The advance of imprinting techniques in biosensor development allowed researchers to standardise the topology of tem-plated cavities to tune sensor selectivity better and bypass the intrinsic limitations of whole protein imprinting.This, in turn, resulted in the possibility of developing precise sensors by solely imprinting the recognition motifs of proteins, known as epitopes, onto polymer structures [119].
The process of epitope imprinting for MIP-based electrochemical sensors has been extensively exploited in literature to use only the recognition sites of proteins as pseudo-templates.This bypasses the hindrances of whole protein imprinting due to bulkiness and the use of short and stable oligopeptide chains for imprinting purposes [120].Epitopes can be obtained through their extraction and isolation from their biological source and faster synthetic approaches, which produce only the peptide chain required for biosensor development instead of the whole protein analyte [121].Moreover, owing to their shorter length than entire proteins, epitopes have predictable primary and/or secondary conformation, unlike complete proteins' complex coiled tertiary structures whose recognition sites may not be adequately exposed for molecular imprinting applications [122].
Nonetheless, epitope imprinting has allowed the development of tailored MIP-based sensors for considerably rare or hard-to-obtain biomarkers and bypassing the imprinting hindrances of whole proteins attributed to size and conformational flexibility [123].In this sense, even when taking into consideration that the contact area required for low-energy binding of whole proteins to receptor cavities ranges between 500-3500 Å 2 as previously commented [100] , the binding of epitopes comprised of oligopeptide chains, short α-helixes and β-strands often exhibit optimal values bellow 2000 Å 2 , thence contributing to their acknowledged selectivity [120] .Furthermore, given the sheer diversity of recognition motifs such as peptides, glycans, monosaccharides, His and FLAG-tags, this technology is upand-coming for biosensing applications [124].
Considering the exposed nature of the binding sites of epitopes and the possibility of achieving optimal electrostatic interactions between these small templates and MIPs, some researchers have exploited this technology to gain very low dissociation constants in protein electroanalysis.For instance, in a recent report, epitopes of human atrial natriuretic peptide underwent detection through a MIP-sensor with a dissociation constant as low as 5.3 μmol l -1 , thereby suggesting outstanding binding affinity and highlighting the possibility of using epitope imprinting for enhancing the sensor performance [125].On the other hand, it must be noted that oligopeptides accounting for up to 30 amino acid units may not be compatible with all polymerisation strategies, and many epitope chains are known to undergo aggregation in aqueous environments.What must also be considered when using these biomaterials in molecular imprinting technologies [120,122].
Their use in crafting sensible and selective MIPbased sensors for electroanalysis has been reported in several fields, ranging from healthcare to foodstuff quality control.For instance, in a recent report, ovalbumin was imprinted on a gold nanoparticle-coated carbon screen-printed electrode, followed by dopamine electropolymerisation.The resulting sensor yielded a meagre detection limit of 10.76 nmol l -1 , reaching a sensibility level of .46parts per million in wine samples [126].Other authors also achieved shallow detection limits by imprinting cytochrome c epitope on poly-3-aminopropyltriethoxysilane; which determinedinute concentrations of this biomarker (i.e., 3.6 ng ml −1 ).Nonetheless, these detection levels were among the lowest reported in the literature, which highlights the effectiveness of epitope imprinting in several applications [127].
Literature states that the imprinting of epitopes can be achieved through grafted or constrained approaches since grafted methodologies are often more reported in developing electrochemical immunosensors, the straightforward embedding of epitopes onto electrode surfaces [128].Grafted epitope imprinting follows the direct modification of the electrode surfaces with the epitope through direct chemisorption or anchoring agents such as SAM-forming units, followed by MIP formation and template removal [128].
On the other hand, constraint epitope imprinting requires the immobilisation of the template on a matrix, followed by MIP formation and removal of both template and matrix.Roving the substrate with the template yields MIP nanoparticles, which can be employed in electrochemical sensing using surface-functionalisation protocols [120].This technology has been used craft MIP nanoparticles that dramatically enhance the analytical performance of immunosensors, and is a promising approach in protein electroanalysis [129].For instance, a recent report showcased the use of cysteine-modified epitopes of neuron-specific enolase as templates in the synthesis of MIP nanoparticles onto gold nanoparticles, thereby yielding an electrochemical sensor with an imprinting factor of 4.2 and a detection range in the picogram level of 25-4000 pg mL -1 [130].
Although epitopes may have limitations for molecular imprinting applications due to possible compatibility issues with solvents and particular morphology, this technology is undeniably an intelligent approach to tackle molecular imprinting while avoiding the drawbacks of wholeprotein imprinting.

CONCLUSIONS
Molecularly imprinted polymer-based electrochemical methods have emerged as a powerful tool for precise protein detection.The amalgamation of molecular imprinting and electrochemical transduction provides a versatile platform with exceptional sensitivity, selectivity, and potential for miniaturisation.Future advancements in this field promise to revolutionise biosensing technologies and foster applications in diverse domains, ultimately contributing to enhanced healthcare, environmental sustainability, and food security.Further research and development in this burgeoning field are imperative to unlock the full potential of MIP-based electrochemical sensors in protein detection.

Figure 1 -
Figure 1 -Schematic of the bulk polymerisation of a monomer-template mixture A -wherein the embedding of the protein template is promoted by the polymerisation of monomers (orange);

Figure 2
Figure 2 depicts how epitope imprinting can selectively detect proteins through their recognition motifs.

Figure 2 -
Figure 2 -Schematic of epitope imprinting technique, wherein the epitope is imprinted in the polymer (orange) A and then removed B. After that, recognising peptides and proteins (blue sphere) is feasible through rebinding the epitope motif C. The epitope structure depicted is the human immunodeficiency virus epitope scaffold, which is stored under the code 3LHP at the Protein Data Bank