Epitope-Imprinted Polymers for Selective Recognition of Bacterial Proteins: From Design to Analytical Applications

Abstract

Epitope-imprinted polymers (EIPs) represent an advanced evolution of molecular imprinting technology for selective bacterial recognition. By using short, surface-exposed peptide fragments derived from bacterial proteins rather than whole cells or full-length proteins, EIPs enable the formation of structurally defined and chemically robust recognition cavities with improved accessibility, stability, and reproducibility. To the best of our knowledge, this review provides the first dedicated and focused overview of EIPs in bacterial detection. The fundamental principles of epitope imprinting are discussed, including rational epitope selection, computational modeling, monomer optimization, imprinting strategies (bulk, surface, nanoMIP, and electropolymerization), and template removal approaches. Representative applications targeting clinically significant pathogens such as Mycobacterium leprae, Salmonella Typhi, and Neisseria meningitidis are critically examined, highlighting analytical performance parameters including detection limits, imprinting factors, selectivity in complex biological matrices, and integration with electrochemical and piezoelectric transducers. In addition to summarizing current achievements, this review evaluates practical limitations related to epitope accessibility, matrix interference, fabrication reproducibility, scalability, and hospital-based implementation. Emerging strategies, including AI-assisted epitope design, multiepitope imprinting, nanomaterial-enhanced architectures, and portable point-of-care systems, are discussed as potential solutions to improve robustness and translational applicability. By consolidating current progress and identifying key scientific and technological gaps, this work clarifies the position of EIPs as promising synthetic recognition elements for next-generation bacterial diagnostics in clinical, food safety, and environmental monitoring.

1. Introduction

Bacterial contamination remains a pervasive and costly global challenge across food safety, clinical diagnosis, and environmental monitoring. Foodborne pathogens continue to cause millions of illnesses annually, threatening public health and disrupting international food trade. In clinical settings, delayed or inaccurate identification of bacterial infections contributes to improper treatment, antimicrobial resistance, and prolonged hospitalization. Similarly, the deterioration of water quality and the rise of antibiotic-resistant bacteria in natural and industrial ecosystems underscore the urgent need for rapid, sensitive, and on-site bacterial detection strategies. These threats necessitate the development of analytical systems for rapid, selective, and reliable pathogen detection.

Conventional laboratory-based techniques, such as culture plating, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), remain the gold standard for bacterial identification. However, despite their sensitivity, these approaches are often time-consuming, labor-intensive, require trained personnel, and depend on costly reagents or cold-chain-unstable biomolecules. Their limitations have significantly motivated the investment in next-generation biosensing platforms that can provide real-time, field-deployable, and cost-effective detection.

Within this context, biosensors have emerged as powerful analytical tools, leveraging biological or biomimetic recognition elements to transduce binding events into measurable signals. Among these, molecularly imprinted polymers (MIPs) stand out as synthetic receptors with extraordinary chemical stability, reusability, low cost, and compatibility with harsh environments, advantages that natural biorecognition elements (e.g., antibodies, enzymes, aptamers) struggle to maintain. − Yet imprinting whole bacterial cells presents inherent difficulties: the large size, complex surface architecture, and fragility of bacterial templates complicate polymerization, limit imprint fidelity, and restrict reproducibility across species, strains, and growth conditions.

To overcome these barriers, the concept of epitope imprinting polymer (EIP) has emerged as a logical and elegant evolution of molecular imprinting technology. Instead of imprinting the entire microorganism, EIP uses short, stable, and structurally defined fragments, such as peptides or cell–surface motifs, derived from bacterial biomarkers. These fragments act as surrogate templates, enabling precise cavity formation that selectively targets the parent bacterium while avoiding the drawbacks of whole-cell imprinting. EIP thus offers a promising route toward highly selective, robust, and reproducible bacterial sensors capable of functioning in complex real samples.

Despite the growing literature on molecular imprinting, the novelty of this review lies in its exclusive focus on EIP sensors for bacterial detection. While several reviews broadly address MIPs for bacteria, − none specifically analyze EIP-driven pathogen sensing. Existing epitope-imprinting reviews tend to center on peptide design, polymerization chemistry, and protein recognition or separation, − without addressing the unique challenges of pathogen detection, nor the translation of EIP into real-sample testing. Conversely, reviews on bacteria-imprinted biosensors overwhelmingly emphasize whole-cell imprinting, mentioning epitope strategies only briefly or tangentially. − Therfore, to the best of our knowledge, this is the first review devoted exclusively to EIP-based bacterial sensors. With the rapid emergence of dual- and multiepitope systems, rational epitope selection, and integration of EIP with electrochemical, optical, and microfluidic devices, a dedicated and application-oriented analysis is urgently needed.

As shown in Scheme , the purpose of this review is to provide a critical and up-to-date analysis of advances in EIP sensors for bacterial detection, emphasizing their underlying design concepts, sensing performance, integration with modern transduction technologies, and effectiveness in complex real samples. In addition to mapping recent progress, we address the key barriers that continue to limit their broader adoption, such as rational epitope selection, control of binding-site architecture, matrix-induced variability, and challenges associated with scalable fabrication, and highlight emerging strategies that aim to resolve these issues. This work clarifies the current landscape, defines key scientific and technological gaps, and outlines promising future directions to accelerate the development of stable, selective, and practical sensing platforms for food safety, clinical diagnostics, and environmental monitoring.

1. Graphical Overview of the Main Contents of This Review.

2. Fundamentals of EIP in MIPs

Fundamentally, molecular imprinting relies on the formation of a polymer network around a target molecule (the template), so that after template removal, complementary binding cavities remain in terms of shape, charge distribution, and functional groups. For low-molecular-weight analytes this strategy is straightforward, but direct imprinting of intact bacteria or even full bacterial proteins is much more problematic. Proteins are large, highly flexible biomacromolecules whose tertiary structure is easily perturbed by organic solvents, extreme pH, or thermal stress, so they tend to denature or become irreversibly trapped within a bulk polymer during imprinting. In addition, complex clinical matrices (serum, plasma, cerebrospinal fluid) introduce “matrix effects” that can mask or distort binding to conventional protein-imprinted films. These issues are even more severe for whole-cell imprinting of bacteria, where the micron-scale cell dimensions, fragile surface structures and biosafety considerations complicate reproducible template handling and cavity formation.

EIP provides a mechanistically sophisticated solution to these challenges by enabling the formation of stable and well-defined recognition sites using short, structurally preserved epitopes (typically 9–15 amino acids), which represent antigenic determinants of bacterial proteins and serve as templates instead of full proteins or whole cells, thereby enhancing selectivity, reproducibility, and compatibility with complex biological matrices. These epitopes are usually chosen to be unique in sequence, solvent-exposed on the native protein surface, and immunorelevant, so that the structural “fingerprint” of the epitope effectively encodes the entire protein in the imprinted cavity. Compared with full proteins, short peptide epitopes used as templates are more robust under the chemical and thermal conditions of imprinting, and avoid unfolding or denaturation of the native structure. They can be synthesized in high purity at relatively low cost and circumvent the difficulties associated with handling bulky, complex protein templates, including their removal and rebinding. As a result, epitope imprinting offers much greater flexibility in the choice of imprinting formats and sensor architectures. Conceptually, epitope imprinting therefore combines the “small-molecule” imprinting mechanism (well-defined, rigid templates and accessible binding sites) with the biological relevance of protein recognition.

In its simplest form, EIP involves selecting a short, antigenically relevant peptide segment that represents a characteristic region of a bacterial surface protein. This epitope is mixed with suitable functional monomers, which self-assemble around the template through noncovalent interactions during polymerization. Once the polymer network solidifies, removal of the peptide leaves behind a complementary binding cavity that preserves the structural features of the epitope. The resulting MIPs can then selectively recognize the same sequence when it appears within the larger native protein or on the bacterial surface, enabling highly specific detection without the need to imprint the entire bacterium. Figure provides a conceptual illustration of EIP process.

1.

Fundamental concept underlying EIP process. Reproduced with permission from ref Copyright 2001 Elsevier.

3. Design Strategy for EIPs

The rational design of EIPs for bacterial sensing begins with the identification of short peptide motifs that faithfully represent structurally accessible and diagnostically relevant regions of bacterial surface proteins. Unlike whole-cell imprinting, where micron-scale cavities are formed that replicate the overall bacterial morphology, EIP generates molecular-scale binding sites complementary to a short, surface-exposed peptide sequence. Consequently, recognition in whole-cell imprinting is primarily governed by surface topology and nonspecific multipoint interactions, whereas EIPs rely on sequence-defined chemical complementarity, enabling antibody-like specificity. This molecular-level precision is the central rationale behind epitope selection, as the chosen peptide must encode a structurally exposed and biologically meaningful recognition determinant capable of guiding the formation of highly defined binding cavities. Figure A illustrates epitope selection strategies. Because bacterial proteins often contain domains that are either buried within the cell envelope or highly variable across strains, epitope selection must prioritize unique, surface-exposed, and sufficiently conserved regions that remain accessible across physiological conditions. In bacterial pathogens such as Neisseria meningitidis, Salmonella Typhi, or Mycobacterium leprae, these epitopes frequently correspond to solvent-exposed loops, immunogenic regions, or flexible C-/N-terminal sequences of outer membrane proteins, invasion factors, or virulence-associated proteins. ,, Selecting such epitopes ensures that the imprinted cavity mimics a biologically relevant recognition determinant and minimizes cross-reactivity with host proteins or commensal bacterial flora.

2.

(A) Selection of target epitopes. (B) Key stages in EIP process. Reproduced from ref Available under a CC BY 4.0 license. Copyright 2021 Teixeira, S. P., Reis, R. L., Peppas, N. A., Gomes, M. E., & Domingues, R. M.

Once candidate epitopes are identified, in-silico modeling provides a powerful predictive tool to refine both the peptide and monomer composition. Docking studies allow simulation of the prepolymerization complex, enabling researchers to choose functional monomers capable of forming stable hydrogen bonding, electrostatic, or π–π interactions with the bacterial epitope. Molecular dynamics simulations further assess the conformational stability of the epitope in the imprinting medium, helping to avoid sequences that may fold, aggregate, or lose their native configuration. These computational strategies, which have been applied extensively in EIP systems targeting bacterial proteins, reduce empirical optimization and guide the creation of cavities that maintain high structural fidelity during rebinding.

From a mechanistic perspective, EIP recognition arises from the interplay between sequence-driven and conformation-driven effects. The selected epitope defines the spatial arrangement of functional groups, enabling sequence-driven recognition through complementary interactions such as hydrogen bonding, electrostatic interactions, and hydrophobic forces, as the amino acid sequence of the template peptide mimics exposed regions of the target protein and dictates binding specificity. At the same time, the three-dimensional arrangement of the epitope during imprinting can be partially encoded within the polymer matrix, introducing a conformation-driven contribution. In protein and epitope imprinting, successful recognition requires preservation of the native-like structure during template formation, as binding sites formed against a specific conformation preferentially recognize the same structural state. Importantly, these two contributions are not independent but inherently coupled, as sequence determines the available interaction motifs while conformation governs their spatial presentation and accessibility. As a result, effective rebinding depends not only on the amino acid sequence but also on the structural compatibility between the imprinted cavity and the epitope as presented in the native protein. This is consistent with the principle of epitope imprinting, where short peptide fragments corresponding to surface-exposed regions enable recognition of the full protein through matching both sequence identity and spatial presentation.

Beyond these mechanistic considerations, a critical practical factor is the accessibility of the selected epitope on intact bacterial cells. While epitope imprinting relies on matching sequence and conformation, effective recognition in real systems requires that the targeted fragment is genuinely exposed and accessible within the native architecture of the bacterial envelope. Computational approaches, including structural modeling (e.g., AlphaFold) and solvent-accessible surface area analysis, can assist in predicting surface-exposed regions, particularly for outer membrane proteins where loop domains are more likely to be accessible. However, such predictions remain inherently approximate and must be experimentally validated, as protein folding, membrane embedding, and the local microenvironment can significantly influence epitope exposure. Techniques such as limited proteolysis (protein shaving) and surface proteomics provide direct experimental evidence of surface accessibility. Importantly, in complex biological environments, epitope accessibility may be further modulated by biofouling, extracellular polymeric substances, or protein corona formation, which can mask surface-exposed binding sites and reduce recognition efficiency. Therefore, integrating computational prediction with experimental validation and testing in relevant matrices are essential to ensure that the selected epitope retains both accessibility and functional relevance for selective recognition.

A variety of polymerization techniques can then be employed to translate the optimized epitope–monomer complex into an imprinted material. Conventional free-radical bulk polymerization remains widely used, particularly for affinity sorbents and hydrogel-type materials, due to its simplicity and compatibility with acrylate/acrylamide chemistries. However, bulk materials suffer from diffusion limitations for large targets, so surface imprinting and thin-film formats are frequently preferred for epitope-based recognition of proteins and pathogens. , Sol–gel chemistries afford inorganic–organic hybrid networks with high rigidity and good chemical stability, and have been successfully combined with epitope templates and fluorescent quantum dots for optical sensing. Electropolymerization offers a major advantage for bacterial sensing because it enables rapid deposition of ultrathin films directly onto transducers such as gold electrodes or quartz crystal microbalance (QCM) while maintaining high site accessibility, an essential factor when recognizing epitopes embedded within large bacterial protein. Meanwhile, nanoMIP or nanogel synthesis, often via solid-phase or emulsion polymerization around immobilized epitopes, produces monodisperse nanoparticles with binding sites confined near the surface, combining fast kinetics with antibody-like affinity. In comparison to microMIPs, nanoMIPs offer significant advantages due to their smaller size and higher surface-area-to-volume ratio. These properties result in faster binding kinetics and increased sensitivity, making nanoMIPs highly effective for detecting bacterial markers, even in low-abundance samples. NanoMIPs are particularly suited for applications requiring miniaturization and rapid detection, such as point-of-care (POC) diagnostics and field-deployable sensors. In contrast, microMIPs, while easier to fabricate and offering greater mechanical stability, often exhibit slower response times and less sensitivity, making them less suitable for quick, sensitive bacterial detection.

Irrespective of format, template removal and rebinding are critical steps that determine the quality of the recognition sites. Short peptide templates are easier to extract than whole proteins and reduce the risk of structural damage to the cavity during elution, but incomplete removal can still cause template bleeding and false-positive signals. Strategies to mitigate this include using milder extraction conditions made possible by the lower molecular weight of epitopes, employing surface-confined or solid-phase imprinting where templates are immobilized on a support and naturally separated from the final MIP nanoparticles, and optimizing cross-linking density so that cavities retain their shape without trapping the peptide irreversibly. During rebinding, structural fidelity of the cavity must be sufficient for the epitope segment within the full protein (or bacterial surface protein) to fit without steric clashes, a requirement that favors surface-accessible epitopes and thin or nanoparticulate formats where binding sites are not buried deep within the polymer bulk. Figure B illustrates the major steps involved in EIP process.

Signal generation in EIP-based sensors originates from the conversion of selective epitope rebinding into a measurable physicochemical change at the polymer/transducer interface. After the target epitope, protein, or bacterial surface marker reoccupies the imprinted cavity, the local interfacial environment is perturbed through changes in surface mass, polymer swelling/shrinking, permeability of the imprinted film, and charge-transfer conditions at the sensing interface. In electrochemical platforms, this rebinding event typically changes the signal of a redox probe or the interfacial charge-transfer resistance because occupation of the cavities can hinder or reorganize probe diffusion and electron-transfer pathways across the imprinted layer. In piezoelectric systems such as QCM/EQCM, recognition is translated into frequency shifts caused primarily by mass uptake on the crystal surface; in liquid-phase measurements, viscoelastic contributions of the adsorbed layer can also influence the response. In optical transducers, target binding alters the optical output by changing the environment of the reporting material or interfacial optical properties, which can be read as changes in fluorescence, plasmonic response, Raman signal, or chemiluminescence. Signal amplification can be further improved by rational material engineering, including ultrathin or surface-confined imprinted films, conductive polymers, nanomaterials, and nanoMIPs with high surface-to-volume ratios and surface-exposed binding sites, all of which improve accessibility of the recognition sites and strengthen coupling between rebinding and signal output. Overall, the sensing mechanism in EIPs can be understood as a stepwise process: selective epitope recognition → interfacial perturbation of the imprinted layer → transducer-dependent signal generation, with the most suitable transduction mode depending on the analytical goal, sample matrix, and device format. Among the available transduction methods, electrochemical techniques are often the most attractive for portable and POC EIP sensors because they are compatible with low-cost, miniaturized, disposable, and field-deployable electrode platforms while maintaining high analytical sensitivity. QCM/EQCM and related piezoelectric methods provide valuable label-free, real-time information on mass rebinding at the sensor surface, but they are more commonly used as benchtop analytical tools than as the simplest portable formats. Optical transduction methods offer high analytical sensitivity and diverse readout formats, including fluorescence, surface plasmon resonance, Raman/surface-enhanced Raman spectroscopy, and chemiluminescence, with strong potential for multiplexed analysis. Nevertheless, depending on the platform design, they may require more sophisticated instrumentation or labeling schemes and may be more vulnerable to background interference in complex biological matrices.

Finally, the performance of EIPs is quantified using parameters analogous to those used for antibodies. Adsorption experiments yield the equilibrium binding capacity (Q) and maximum capacity (Q max), while comparison of MIP and nonimprinted polymer (NIP) responses provides an imprinting factor that reflects the contribution of the imprinted cavities. Binding isotherms and surface-based techniques such as QCM or surface plasmon resonance are used to determine apparent dissociation constants (K _D) and kinetic rate constants, giving a measure of binding affinity and association/dissociation kinetics. Selectivity is evaluated by challenging the EIP with structurally related peptides, mutated epitopes, or homologous proteins and calculating selectivity coefficients or cross-reactivity ratios. , In addition, long-term stability, chemical, thermal, and mechanical, is assessed under relevant storage and operating conditions; one of the major advantages repeatedly underlined for EIPs is their robustness compared with protein antibodies, including tolerance to organic solvents and wider pH and temperature ranges.

4. Applications in Bacterial Detection

EIP has emerged as a powerful strategy for developing highly specific and robust bacterial sensing platforms. By creating synthetic recognition sites that mimic the spatial and chemical features of key bacterial epitopes, molecularly imprinted materials can discriminate between closely related species and even distinguish strains based on subtle molecular signatures. This approach overcomes several limitations of traditional whole-cell imprinting, offering enhanced stability, easier template preparation, and improved selectivity toward clinically or environmentally relevant bacterial markers. As a result, epitope-imprinted sensors have been successfully integrated into diverse detection formats, from electrochemical and optical systems to portable field-deployable devices, supporting rapid, sensitive, and cost-effective bacterial monitoring. These advantages make EIP a promising platform for addressing challenges in diagnostics, food safety, biodefense, and environmental surveillance. The following sections highlight major bacterial targets and demonstrate how epitope imprinting has been adapted to detect different pathogens with high precision. Table provides comprehensive summary of EIP-based bacterial sensing, including representative examples for M. leprae, S. typhi, and N. meningitidis. The table illustrates how the choice of epitope, monomer composition, and polymerization format dictate analytical performance.

1. EIP Sensors for Bacterial Detection in Human Samples: Design Strategies, Detection Modes, and Analytical Performance Comparison.

bacterial target	epitope identity	monomer	polymerization format	detection technique	imprinting factor	analytical performance	sample	references
M. leprae	LDIYTTLARDMAAIP (LP-15)	3-sulfopropyl methacrylate potassium salt, Benzyl methacrylate, and 4-aminothiophenol	Electropolymerization	EQCM	8.28	LOD: 0.161 nM; LOQ: 0.536 nM; RSD: 2.01%; Working range: 10–140 nM	Human blood
S. typhi	WC-15 and NC-15	2-methacryloy loxyethyl phosphorylcholine, methacrylic acid, and benzyl methacrylate	Electropolymerization	EQCM	WC-15:6.93 NC-15:7.00	LOD: WC-15:1.65 nM, NC-15:0.025 nM; LOQ: WC-15:5.03 nM, NC-15:0.075 nM; Working range: 100–1000 nM	Human blood
S. typhi	WTC-15	Methacrylic acid, 2-methacryloyloxyethylphosphorylcholine, and Benzyl methacrylate	Bulk copolymerization	EQCM and DPV	EQCM: 14.4	EQCM: LOD: 0.52 nM; LOQ: 1.7 nM	Human blood
					DPV: 8.53	DPV: LOD: 0.16 nM; LOQ: 0.54 nM; RSD: 4.6%; Working range: 6–90 nM
N. meningitidis	KPYAKNSVALQAVC	3-sulfopropyl methacrylate potassium salt and benzyl methacrylate	Bulk polymerization	EQCM	12.27	LOD: 1.2 nM; Working range: 0–20 nM	Human blood
N. meningitidis	CGRHNSESYH	3-thiophene acetic acid	Electropolymerization	EQCM and DPV	3.84	LOD: 8.4 nM; Working range: 4.4–40 nM	Human blood serum
N. meningitidis	KGLVDDADI	Methacrylic acid	Thermal polymerization	EQCM	3.34	LOD: 15 nM; Working range: 97 nM	Human blood serum
N. meningitidis	CH-10: CGRHNSESYHKC-10: KGLVDDADIC	3-sulfo propyl methacrylate potassium salt and benzyl methacrylate	Electropolymerization	EQCM	CH-10:14.72, KC-10:10.15	LOD: CH-10:0.68 nM, KC-10:1.01 nM	Human blood serum
						LOQ: CH-10:2.05 nM, KC-10:3.05 nM
						RSD: CH-10:5.02%; KC-10:4.84%; Working range: 100–1000 nM

4.1. Mycobacterium leprae

Mycobacterium leprae (M. leprae) is an obligate intracellular pathogen and the causative agent of leprosy, a chronic infectious disease that can remain silent for years before clinical symptoms appear. Its incubation period may extend up to a decade, and in many patients the bacterial load is initially very low, which makes early diagnosis particularly difficult. Conventional serological tests, especially those based on antibodies against the characteristic phenolic glycolipid-I of the cell wall, often lack sensitivity in paucibacillary forms of the disease. As a result, a substantial proportion of infections remain undetected until nerve damage and deformities are already established, despite global efforts to reduce leprosy burden and achieve early case detection. These diagnostic limitations have driven the search for more specific and sensitive tools that can identify M. leprae infection at an early or even subclinical stage. In this context, Kushwaha et al. fabricated an EIP nanoparticle film on a gold-coated EQCM electrode for selective detection of M. leprae. A computationally identified surface epitope (LP-15: LDIYTTLARDMAAIP) was used as the template and imprinted using a multimonomer strategy. The polymeric adduct nanoparticles were synthesized in bulk, repeatedly ultrasonicated and centrifuged, then electrodeposited onto the gold EQCM surface via electropolymerization. Subsequent template extraction generated surface-accessible cavities capable of selective rebinding (Figure A). Atomic force microscopy (AFM) characterization of the modified electrodes revealed particle sizes predominantly in the range of 200–300 nm, with average sizes varying from 209 to 276 nm. The MIP film exhibited a rough surface morphology featuring numerous cavities, whereas the NIP filmprepared under identical electropolymerization conditions, differing only in the absence of the epitope templatedisplayed a comparatively smoother surface (Figure B). The sensor withstood comprehensive analytical tests, including selectivity, specificity, matrix effect, reproducibility (RSD 2.01%), and achieved an imprinting factor (IF) of 8.28 over a working range of 10–140 nM, with a detection limit (LOD) of 0.161 nM and a quantification limit (LOQ) of 0.536 nM. The study demonstrates that imprinting short, pathogen-specific peptide sequences can confer exceptional selectivity, enabling discrimination not only against plasma proteins but also against closely related peptide analogues differing by only a few amino acids. The achieved subnanomolar detection limit and successful validation in infected patient blood samples highlight the analytical strength of EIP for early stage bacterial diagnosis. However, the reliance on multimonomer synthesis, electropolymerization, and EQCM instrumentation increases system complexity, and IF, derived from response comparison at defined analyte concentrations rather than calibration sensitivity, may limit the robustness of performance evaluation. These aspects suggest that while this approach is highly effective for proof-of-concept clinical sensing and mechanistic validation, further simplification and standardized performance assessment will be required before translation into scalable or POC bacterial diagnostic platforms.

3.

(A) Schematic of the LP-15 epitope-imprinted EQCM fabrication process. (B) AFM images show: (a) MIP nanoparticles; (b) NIP nanoparticles; (c) an EQCM electrode coated with MIPs; and (d) an EQCM electrode coated with NIPs. Reproduced with permission from ref Copyright 2019 Elsevier.

4.2. Salmonella Typhi

Salmonella enterica serovar Typhi (S. typhi) is the causative agent of enteric (typhoid) fever, an acute systemic infection that remains a major public-health concern in many developing countries. Clinically, typhoid often presents as a nonspecific febrile illness that is difficult to distinguish from other common infections such as malaria and dengue, especially in regions where these diseases cocirculate. Conventional blood culture suffers from limited sensitivity and is strongly affected by factors like prior antibiotic use and inconsistent access to culture facilities. Serological tests can also yield misleading results, as highlighted by false-positive typhoid or dengue tests reported in COVID-19 patients, further complicating diagnosis in endemic settings. These limitations create an urgent need for sensitive, selective, and rapid detection strategies for S. typhi infection. To address this, Singh et al. engineered a single-epitope (WTC-15) MIP sensor targeting a SipD-derived peptide from S. Typhi, integrated with an EQCM transducer (Figure a). AFM characterization revealed compact and uniform coverage of both the MIP and NIP films on the gold electrode surface, with distinct morphological differences (Figure b). The MIP film (Figure b (A&B)) exhibited a rough surface containing well-defined imprinted cavities formed after WTC-15 extraction, whereas the NIP film (Figure b (C&D))prepared under identical conditions and also subjected to the extraction solventlacked such cavities and appeared considerably smoother, indicating successful generation of selective recognition sites. Quantitative analysis confirmed this, with a root-mean-square roughness of 98.967 nm for the MIP versus 12.45 nm for the NIP, demonstrating high cavity fidelity. These morphological features contribute directly to the sensor’s high selectivity and near-homogeneous binding. The sensor exhibited high selectivity toward the imprinted epitope and enabled specific, selective detection in real samples from typhoid-infected patients. Over a working range of 6–90 nM, the sensor achieved IF of 8.53 (DPV) and 14.4 (piezoelectric measurements). LODs were 0.16 nM (DPV) and 0.52 nM (piezoelectric), with corresponding LOQs of 0.54 nM and 1.7 nM, respectively. The single-epitope strategy provided a wide linear range and homogeneous binding sites (Freundlich m = 1), which are advantageous for quantitative peptide assays, though it does not capture the full bacterial protein as dual-epitope systems can. The polymerization method further reinforced this performance. Bulk polymerization with electrodeposition, using ethylene glycol dimethacrylate (EGDMA) as the cross-linker, produced a rough MIP surface that enhances cavity accessibility. The inclusion of zwitterionic 2-methacryloyloxyethyl phosphorylcholine minimized nonspecific adsorption, ensuring reliable detection in complex biological matrices such as blood plasma. Regarding transduction, the EQCM-only platform enables sensitive detection without the additional complexity of nanocomposite fabrication. While precise control of polymer deposition and measurement conditions is required, this approach achieves excellent LODs and IFs, making it suitable for high-specificity applications when whole-pathogen capture is not necessary. Despite these strengths, practical limitations remain. IF was calculated from the ratio of signal responses (ΔI or ΔF) between the imprinted and nonimprinted polymers at defined analyte concentrations, rather than from calibration sensitivity (slope) comparison, which may reduce robustness and cross-study comparability. Additionally, the requirement for EQCM instrumentation and carefully controlled fabrication and measurement conditions limits adaptability to simpler or high-throughput workflows. Overall, this system functions effectively as a high-specificity benchmark for epitope-based bacterial recognition, illustrating the advantages of immunoinformatics-guided epitope selection and multimonomer imprinting in creating reliable, selective MIP sensors. Building on this approach, Srivastava et al. advanced the EIP concept by introducing dual-epitope imprinting of SipD combined with an EQCM transducer and a conductive AuNP–MoS₂ scaffold (Figure c). Transmission electron microscopy (TEM) characterization confirmed the successful synthesis of the nanocomposite (Figure d). The MoS₂ nanosheets displayed a hexagonal, thin, wrinkled morphology, with in situ growth of gold nanoparticles uniformly distributed across their surfaces (Figure d­(a)). The well-dispersed AuNPs had an average diameter of 15 nm, confirming effective integration of the nanoparticles onto the MoS₂ scaffold (Figure d­(b)). In this study, the dual-epitope design produced heterogeneous binding sites (Freundlich m = 0.249–0.477) and a narrower linear range (100–1000 nM), but allowed capture of the whole S. Typhi protein, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a capability not achievable with single-epitope systems. Bulk polymerization with electrodeposition using N,N-methylene-bisacrylamide as cross-linker generated a more hydrophilic MIP surface (contact angle 54.5°) compared to EGDMA, further reducing nonspecific adsorption. The EQCM–AuNP–MoS₂ platform provided electrochemical amplification, achieving an ultralow LOD compared to single-epitope EQCM sensors. Performance metrics included LODs of 1.65 nM and 0.025 nM, LOQs of 5.03 nM and 0.075 nM, and IFs of 6.93 and 7.00 for WC-15 and NC-15, respectively, with negligible matrix interference in untreated blood plasma. Despite these advantages, IF was calculated from response ratios (Δf _MIP/Δf _NIP) rather than calibration slopes, limiting reliability and consistency across studies. Additionally, the complexity of docking-guided multimonomer synthesis, nanostructured electrode fabrication, and EQCM instrumentation may restrict translation to simplified POC platforms.

4.

(a) Illustration showing the fabrication steps of the epitope-imprinted polymer on the gold EQCM electrode. (b) AFM 2D and 3D images show: MIP (A,B) and NIP (C,D), respectively. Reproduced from ref Available under a CC BY 4.0 license. Copyright 2024 Singh, R., Nair, M. L., Raghuwanshi, R., Nath, G., & Singh, M. (c) Schematic illustration of the fabrication steps for the dual-epitope imprinted polymer EQCM sensor. (d) TEM images depict: (a) AuNPs-MoS₂NSs composites, illustrating the hexagonal structure of MoS₂NSs, and (b) a higher-resolution image of the AuNPs-MoS₂NSs. Reproduced from ref Available under a CC BY 4.0 license. Copyright 2025 Srivastava, A., Kushwaha, A. K., Sagar, P., Parida, A., Singh, R. S., Srivastava, S. K., Raghuwanshi, R., Nath, G.,& Singh, M.

4.3. Neisseria meningitidis

Neisseria meningitidis (N. meningitidis) is a major cause of bacterial meningitis and septicemia and has been highlighted by WHO as a pathogen capable of triggering large-scale epidemics. Early infection often presents with nonspecific symptoms and can be easily misdiagnosed, while many existing diagnostic methods (culture, PCR, latex agglutination, antigen tests) typically identify the disease only at an advanced stage, when the bacteria have already disseminated in the host. This delay in reliable diagnosis is linked to high mortality and severe neurological sequelae, underscoring the need for rapid, sensitive tools that can detect meningococcal infection at an early stage, ideally directly from patient samples without complex pretreatment. In response to this, Gupta et al. demonstrated epitope imprinting of an outer membrane protein of N. meningitidis using a computationally selected peptide epitope, showing that peptide-level imprinting can selectively recognize the native protein directly in infected human serum without extensive pretreatment. In this study, the single-epitope strategy produced homogeneous binding sites, evidenced by a single oxidation peak during electropolymerization, and a linear working range of 4.4–40 nM. While this range is adequate for detecting the target protein, it may be less robust against epitope variants than a dual-epitope approach. The polymerization methodelectropolymerization of 3-thiophene acetic acid directly on a self-assembled monolayer of the epitopeeliminated the need for separate cross-linkers or initiators and generated an ultrathin imprinted film (4–5 nm by AFM) with free carboxyl groups for analyte interaction, although this simpler chemistry contributed to a modest IF of 3.84. The transduction platform integrated EQCM with DPV using a K₃[Fe­(CN)₆] redox probe, providing dual piezoelectric and electrochemical readout without nanomaterial amplification. This kept fabrication straightforward but resulted in LOD of 8.4 nM, which is less sensitive than platforms employing nanostructured scaffolds, yet remains clinically relevant for early diagnosis of meningococcal disease. The key strength of this study lies in establishing that a single, well-chosen epitope can effectively encode protein specificity while minimizing nonspecific binding from abundant plasma proteins, addressing a central bottleneck in bacterial diagnostics. At the same time, dependence on a single epitope as the recognition motif may limit robustness toward protein variants or partially masked epitopes. Additionally, the IF was calculated from a single-point binding response rather than from calibration slope comparison, which may overestimate selectivity under varying analyte concentrations. Overall, this approach validates epitope imprinting for Neisseria detection but suggests that more elaborate strategies, such as dual-epitope designs or multimonomer matrices, may be required to ensure reliable recognition under biologically heterogeneous conditions. In contrast, Gupta et al. advanced epitope-imprinted bacterial sensing by targeting the iron-binding protein of N. meningitidis through a multiple-monomer imprinting strategy integrated on an EQCM platform (Figure ). Rather than relying solely on epitope selection, this approach emphasizes chemical refinement of the imprinted binding environment, enabling multipoint noncovalent interactions, electrostatic, hydrophobic, and π–π, that significantly enhance binding strength and improve discrimination of the native protein in infected serum samples. Regarding the effects of design choices on performance in this study, the single-epitope strategy (using a 13-mer sequence KPYAKNSVALQAVC from fbpA) produced highly specific recognition with an IF of 12.27, demonstrating that a well-chosen epitope can achieve strong binding when combined with multiple complementary monomers. The polymerization method, bulk polymerization with two functional monomers (3-sulfopropyl methacrylate potassium salt and benzyl methacrylate) plus a cross-linker (N,N′-Methylenebisacrylamide), generated an optimized multipoint binding environment, though the added complexity increases synthetic demands and requires careful optimization. The EQCM transduction platform provided real-time, label-free mass detection with nanogram sensitivity (LOD of 1.2 nM), enabling direct monitoring of epitope binding and extraction without fluorescent or redox labels. Unlike the previous study, which combined EQCM with DPV for dual piezoelectric and electrochemical readout, this simpler EQCM-only approach avoids additional instrumentation and fabrication steps, facilitating rapid diagnostic development. However, the absence of electrochemical amplification may limit sensitivity for very low analyte concentrations, a limitation partially mitigated by the high-affinity multipoint imprinted matrix. While the elevated IF and lower LOD underscore the benefits of multimonomer design in mitigating the recognition fragility inherent to simpler epitope systems, it should be noted that the reported IF was calculated from single-point binding measurements rather than from a comparison of full calibration curve slopes. Consequently, this method may overestimate selectivity across the complete concentration range.

5.

Synthesis scheme for epitope-specific MIPs via combined epitope and surface imprinting. Reproduced with permission from ref Copyright 2018 Elsevier.

5. Therapeutic Applicability of EIPs with Natural and Synthetic Receptors

The development of EIPs has opened up new avenues for therapeutic applications, providing a synthetic alternative to natural receptors like antibodies and enzymes, and synthetic receptors such as aptamers and synthetic antibodies. While natural receptors offer unmatched specificity and affinity, their limitations include instability under nonphysiological conditions, high production costs, and challenges in reusability. − Synthetic receptors, on the other hand, offer advantages such as tailored design, high stability, and ease of production, but they often face challenges in matching the binding affinity of natural counterparts. −

EIPs bridge this gap by enabling selective recognition of short, surface-exposed bacterial epitopes without the need for whole-cell imprinting, providing robust performance in complex biological matrices, high reusability, and cost-effective production. ,, Importantly, their compatibility with electrochemical and QCM-based platforms makes them highly suitable for POC bacterial detection, particularly at low pathogen loads. , Nevertheless, their performance remains dependent on epitope selection and monomer design, with potential limitations in strain-level discrimination and biocompatibility that must be carefully optimized for clinical and in vivo applications. , Table illustrates the key performance trade-offs between EIPs, natural receptors, and synthetic receptors, emphasizing their distinct recognition mechanisms, analytical capabilities, operational stability, and practical applicability in bacterial diagnostics.

2. Comparative Features of EIPs with Natural Receptors and Synthetic Receptors in Bacterial Recognition.

feature	EIPs	natural receptors	synthetic receptors	references
bacterial recognition strategy	recognition of short, surface-exposed peptide epitopes that encode bacterial protein identity without imprinting whole cells	recognition of full antigen via evolved 3D antigen–antibody interfaces	recognition of purified protein or whole-cell targets through in vitro selection or engineering	,,
strain-level discrimination	high when epitope sequence is unique and conserved; limited if epitope variability exists across strains	very high; capable of distinguishing closely related strains	high, especially with well-optimized aptamers; may require reselection for new strains	,,
sensitivity for low bacterial load detection	good to excellent when integrated with electrochemical or mass-sensitive platforms	excellent due to biological amplification mechanisms	high; depends on signal amplification strategy	,,
performance in complex biological matrices (blood, serum, food)	robust polymer matrix resists denaturation; susceptible to surface fouling but chemically stable	sensitive to denaturation and matrix-induced conformational changes	generally stable; nucleic-acid aptamers may degrade in biological fluids without modification	,,
reusability in bacterial detection cycles	high; binding sites remain functional after multiple regeneration steps	generally low; activity decreases after regeneration	variable; aptamers can be reusable with proper regeneration	,,
cost considerations for bacterial diagnostics	relatively low-cost polymer chemistry; no need for cold-chain storage	high production and storage costs; cold-chain required	moderate; synthesis cost decreases after sequence identification	,,
biocompatibility and in vivo applicability	depends on monomer choice; traditional methacrylate systems may raise toxicity concerns	inherently biocompatible but may provoke immune response	generally good biocompatibility; depends on chemical modification	,,
suitability for POC bacterial detection	highly compatible with portable electrochemical and QCM-based platforms; no biological fragility	often requires careful immobilization to maintain activity	suitable for miniaturized biosensors; may require nuclease protection	,,
Batch-to-batch reproducibility	high theoretical reproducibility due to controlled polymer synthesis; dependent on imprinting conditions	biological variability between production batches	high reproducibility for chemically synthesized aptamers	,,

6. Analytical and Practical Challenges of Epitope-Imprinted Sensors

Epitope-imprinted sensors show strong potential for selective bacterial detection, but several analytical and practical barriers still limit their wider use. Key concerns include epitope selection, template removal, matrix interference, template orientation, reproducibility, clinical adoption, and material safety, as summarized in Scheme .

2. Analytical and Practical Challenges of Epitope-Imprinted Sensors.

6.1. Epitope Selection

The selection of an appropriate epitope is a central determinant of successful bacterial recognition using EIPs. Because EIPs reproduce only a short peptide fragment rather than the entire bacterial cell or full-length surface protein, the chosen epitope must accurately represent a region that is accessible on the native bacterium under realistic assay conditions. This creates a fundamental distinction between peptide-level recognition, which is primarily governed by sequence-specific interactions under controlled conditions, and full-protein recognition, which additionally depends on the accessibility and native structural context of the epitope within the intact biomolecule. If the selected sequence is buried within the tertiary structure of the protein, masked by other membrane components, or sterically hindered on the bacterial surface, the imprinted cavity may exhibit strong binding toward the synthetic peptide during calibration but fail to recognize the intact bacterial target in complex samples. For bacterial sensing applications, epitope accessibility must therefore be evaluated in the context of the whole cell envelope architecture, particularly in Gram-negative organisms where outer membrane organization, lipopolysaccharide layers, and protein–protein interactions may partially obscure candidate sequences. In addition, strain-to-strain variability poses a critical challenge. Epitopes that are not sufficiently conserved across clinically or environmentally relevant strains may result in selective detection of only specific variants, limiting diagnostic robustness and field applicability. Conversely, epitopes with high homology to proteins from commensal or closely related bacterial species may compromise selectivity. Another important consideration is the structural behavior of the peptide template during imprinting. Isolated peptide fragments may adopt conformations that differ from their native presentation within the full-length protein, potentially leading to cavities that do not optimally accommodate the epitope in its biological context. Ensuring that the selected epitope maintains structural relevance to the native bacterial protein is therefore essential to achieve reliable and reproducible bacterial detection rather than merely peptide recognition. Importantly, this highlights a fundamental distinction between epitope-level recognition and full-protein binding. While EIPs are engineered to recognize a specific peptide sequence, successful detection of the native protein depends on the preservation of that epitope’s accessibility and structural context within the intact biomolecule. In this regard, such conformational mismatch between the isolated peptide template and its native presentation in the full-length protein may arise, potentially affecting binding performance. ,, As a result, binding performance toward isolated peptides may overestimate sensor performance unless these factors are explicitly accounted for during epitope selection and imprinting design.

6.2. Template Removal

Efficient template removal is a decisive step in ensuring reliable bacterial recognition by EIPs. Because EIPs are designed to detect epitopes embedded within native bacterial surface proteins, any residual peptide remaining in the polymer matrix can directly compromise sensor performance. Incomplete removal leaves occupied binding sites that reduce the effective number of accessible cavities, thereby limiting sensitivity toward the target bacterium in real samples. An additional concern in bacterial sensing is template bleeding, where traces of the synthetic epitope leach from the polymer during measurement. In electrochemical or mass-sensitive platforms such as EQCM, released peptide fragments may generate false signals or artificially enhance apparent binding responses. This effect is particularly problematic when detecting low bacterial loads in clinical or environmental matrices, where analytical accuracy is critical. Template extraction must also preserve the structural fidelity of the imprinted cavities. Harsh removal conditions, including strong solvents or extreme pH, may distort the cavity architecture (so-called “memory collapse”), reducing its complementarity to the epitope as presented within the intact bacterial protein. Since bacterial detection relies on recognition of the epitope in its native biological context rather than as an isolated peptide, even subtle cavity deformation can impair selectivity and binding efficiency. Therefore, achieving complete peptide removal while maintaining cavity integrity remains a key practical challenge in translating epitope imprinting from peptide-level recognition to robust and reproducible detection of whole bacterial targets in complex samples.

6.3. Matrix Interference

Matrix interference represents a major limitation in translating EIPs from controlled laboratory conditions to practical bacterial detection in complex samples such as blood, serum, or food extracts. In real matrices, the imprinted surface is exposed not only to bacterium surface proteins but also to a wide range of nonspecific components, including abundant proteins (e.g., albumin, globulins), lipids, polysaccharides, cellular debris, and inorganic ions. These species can adsorb onto the polymer surface or within the pores of the imprinted layer, partially blocking access to recognition cavities that are intended to bind bacterial epitopes. Such fouling effects are particularly problematic when detecting low-abundance surface proteins, as nonspecific adsorption may mask the relatively weak and localized epitope–cavity interactions. In electrochemical and mass-sensitive platforms, matrix-induced surface changes can alter baseline signals, reduce sensitivity, and increase variability, thereby compromising accurate quantification of bacterial load. Furthermore, variations in pH, ionic strength, and the presence of divalent ions in food or environmental samples can weaken hydrogen bonding or electrostatic interactions that stabilize epitope recognition, diminishing selectivity toward the target bacterium. Another important consideration is cross-reactivity in polymicrobial or microbiota-rich environments. Structurally similar proteins from nontarget or commensal bacteria may compete for binding, especially if the selected epitope shares partial sequence homology. Therefore, ensuring reliable bacterial recognition in complex matrices requires not only high imprinting fidelity but also strategies to minimize nonspecific adsorption and maintain selective epitope accessibility under realistic sample conditions.

6.4. Challenges of Template Orientation in EIPs

A significant limitation in the design of EIPs is the lack of controlled orientation of the peptide template during the imprinting process. In traditional imprinting workflows, the epitope peptide is simply dissolved in the prepolymerization mixture, which results in random orientations of the template relative to the growing polymer network. This random presentation can lead to heterogeneous binding sites with variable functional group exposure, reducing the uniformity of the resulting cavities and ultimately diminishing both affinity and selectivity toward the target region of the protein. Such heterogeneity is particularly problematic when the binding site must recognize a precise spatial arrangement of functional groups on the native protein surface. Without control over the template orientation, a significant fraction of the binding cavities may fail to faithfully reproduce the geometry required for high-fidelity recognition. Moreover, random template orientations can result in partial or buried binding motifs within the polymer matrix, hindering efficient template removal and subsequent target rebinding, and increasing nonspecific interactions that complicate analytical performance. This challenge of uncontrolled template orientation and the resulting site heterogeneity has been recognized as an intrinsic drawback of bulk epitope imprinting and a major factor limiting recognition efficiency in EIPs, particularly for larger or structurally complex protein targets.

6.5. Reproducibility and Scalability

Reproducibility and scalable fabrication remain critical barriers to the practical deployment of EIPs for bacterial detection. Because successful bacterial recognition depends on the formation of well-defined and accessible binding cavities that accurately match surface-exposed epitopes, even minor variations in synthesis parameters can significantly affect sensor performance. Small deviations in monomer composition, cross-linking density, polymerization kinetics, or epitope orientation may alter cavity geometry and surface accessibility, leading to batch-to-batch variability in sensitivity, selectivity, and imprinting factor. In bacterial sensing platforms, additional sources of variability arise from electrode surface preparation, nanomaterial integration, electropolymerization conditions, and template immobilization efficiency. Since many reported EIP-based bacterial sensors rely on surface-confined thin films or nanoparticle assemblies, slight inconsistencies in film thickness or nanoparticle distribution can influence epitope accessibility and mass-transfer properties, ultimately affecting the ability to detect intact bacterial proteins in complex matrices such as blood or food extracts. These challenges become more pronounced when transitioning from proof-of-concept laboratory fabrication to large-scale production. Maintaining uniform reaction environments, consistent peptide quality, and standardized extraction protocols is more difficult at scale, increasing the risk of performance drift. Moreover, manual procedures, such as peptide immobilization, washing steps, or template removal, may introduce operator-dependent variability that complicates standardization and regulatory validation. For EIP-based bacterial sensors to move toward commercialization and routine diagnostic use, fabrication protocols must therefore be simplified, automated, and tightly controlled to ensure reproducible bacterial recognition across batches and production scales.

6.6. Challenges for Hospital-Based Adoption

For hospital-based adoption, EIP-based sensors must be benchmarked against established methods such as culture, PCR, and ELISA with respect to the main criteria that define clinical applicability. Culture remains indispensable because it detects viable organisms, supports antimicrobial susceptibility testing, and is interpreted against established clinical thresholds in colony forming unit (CFU)/mL; however, it is slow, typically requiring 24–72 h, and although consumable costs are relatively low, it is labor-intensive and less suitable for rapid front-end screening. PCR-based assays generally provide the highest analytical sensitivity and are best suited to very low-burden infections, with many rapid molecular platforms reporting results in less than 2 h, but they depend on specialized instrumentation, trained personnel, and relatively high operational cost. ELISA and related immunoassays are usually less analytically sensitive than nucleic-acid amplification and their clinical performance depends strongly on antigen abundance and antibody quality, but they remain attractive in hospitals because they can be completed within hours rather than days and are readily adapted to batch processing and automated high-throughput workflows. In comparison, currently reported EIP-based bacterial sensors show laboratory limits of detection of approximately 0.025–15 nM for bacterial epitopes or proteins in blood or serum, indicating promising analytical sensitivity, ,,,, but these values are still largely expressed for purified epitopes/proteins rather than mapped to standardized clinically actionable thresholds such as CFU/mL-based cut-offs used in routine microbiology. In terms of time-to-result, EIPs are attractive because they are commonly integrated with electrochemical or piezoelectric transducers that enable rapid signal acquisition and, in some cases, near-real-time readout once binding occurs, making them fundamentally faster than culture and potentially competitive with rapid front-end screening formats. Their main advantage in cost and scalability lies in the use of low-cost synthetic polymers and short peptide templates, together with high physicochemical stability, no intrinsic cold-chain requirement, and compatibility with miniaturized sensing platforms; however, unlike culture, PCR, and ELISA, EIPs still lack standardized manufacturing, interlaboratory validation, and clinically established threshold frameworks. ,, Thus, the most realistic near-term role of EIP-based sensors is not necessarily to replace PCR in absolute sensitivity or culture in viability-based diagnosis, but to provide rapid, stable, and potentially lower-cost complementary tests, provided that future studies translate their favorable laboratory detection limits into clinically relevant thresholds in real patient specimens.

6.7. Challenges in Toxicity, Biocompatibility, and Biodegradability

While EIPs have demonstrated considerable potential in bacterial sensing and diagnostic applications, their use in in vivo environments presents significant challenges, including toxicity, biocompatibility, and biodegradability. Despite showing promising binding performance in vitro, EIPs often experience reduced recognition ability in vivo due to factors such as matrix interference, residual template presence, limited stability in biological environments, and immune responses. Addressing these issues is crucial for the successful application of EIPs in clinical diagnostics, therapeutic treatments, and long-term implantation in living organisms. EIPs, being synthetic materials, carry potential toxicity risks due to their monomers or cross-linkers when used in in vivo environments. Methacrylate-based polymers, commonly used in EIP fabrication, can be cytotoxic at certain concentrations, potentially leading to cell death or organ toxicity if not properly controlled. The toxicity of residual monomers or degradation products must be evaluated in in vivo models, ensuring that these materials are nontoxic to humans or animals. In addition to toxicity, EIPs must exhibit biocompatibility, the ability to interact with biological tissues without causing harm. This includes ensuring that EIPs do not elicit immune responses, which could lead to inflammation, rejection, or chronic infection. The surface properties of EIPs may need to be modified to improve their biocompatibility, particularly in reducing protein adsorption or immune system activation. A critical challenge for in vivo applications is the biodegradability of the EIPs. Nonbiodegradable polymers can accumulate in the body over time, leading to potential toxicity and other adverse effects. To ensure EIPs do not persist indefinitely in the body, they must degrade into nontoxic byproducts that can be safely eliminated.

7. Emerging Trends and Future Directions

Emerging trends in epitope-imprinted sensors are directed toward overcoming current limitations while improving selectivity, stability, and clinical applicability. In particular, growing attention is being given to artificial intelligence (AI)-assisted epitope design, machine learning (ML)-guided monomer selection, hybrid nanomaterial-based EIPs, sustainable and biodegradable materials, multiepitope imprinting, advanced surface modification, better control of template orientation, and the integration of portable POC diagnostic systems, as summarized in Scheme .

3. Emerging Trends and Future Directions in Epitope-Imprinted Sensors.

7.1. AI-Assisted Epitope Design and Machine Learning-Driven Monomer Screening

An important emerging direction in the development of EIPs for bacterial detection is the integration of AI and ML to rationalize epitope selection and monomer formulation beyond empirical trial-and-error approaches. In the context of bacterial sensing, relevant input data are increasingly accessible, including annotated bacterial proteomes, experimentally validated virulence factors, antigen databases, and high-confidence structural predictions. These resources enable AI-driven pipelines to prioritize candidate epitopes based on criteria directly linked to successful bacterial recognition, such as surface exposure on intact cells, conservation across clinically relevant strains, minimal homology to host proteins, and predicted structural stability under physiological conditions. For pathogen detection, the objective is not merely strong peptide binding, but reliable recognition of the epitope as presented within the native bacterial surface protein. Accordingly, computational models must evaluate parameters that influence accessibility in the full cell-envelope context, particularly in Gram-negative bacteria where outer membrane organization, lipopolysaccharides, and protein–protein interactions may partially mask candidate sequences. Predictive workflows can therefore incorporate structural modeling of membrane-embedded proteins and simulate epitope exposure under realistic environmental conditions. On the materials side, ML-assisted screening of functional monomers can optimize polymer composition for bacterial sensing applications. Instead of relying solely on theoretical binding affinity to isolated peptides, models can rank monomer combinations based on descriptors relevant to imprinting performance, such as multipoint interaction potential, resistance to template entrapment, stability in physiological ionic strength, and predicted cavity robustness during repeated bacterial binding cycles. By narrowing the experimental design space, these approaches may enhance reproducibility and reduce fabrication variability, thereby strengthening the reliability of EIP-based bacterial sensors. Nevertheless, several limitations must be acknowledged in the context of pathogen detection. Available training data sets are often biased toward well-characterized bacterial species, increasing the risk of overfitting and limiting generalizability to emerging or less-studied pathogens. In addition, epitopes predicted to be solvent-exposed in silico may become partially inaccessible in vivo due to conformational rearrangements, strain-specific mutations, or interactions with host factors. Rapidly evolving bacterial pathogens further complicate model robustness, particularly when targeting variable virulence proteins.

7.2. Development of Hybrid EIPs with Nanomaterials

The integration of nanomaterials into EIPs represents a promising strategy to enhance the analytical performance of bacterial detection platforms. Nanostructured materials such as graphene oxide, metal–organic frameworks, quantum dots, carbon nanotubes, and metal nanoparticles provide high surface area, improved electrical conductivity, and versatile surface functionalization, all of which are advantageous for amplifying signal transduction in electrochemical, optical, or mass-sensitive bacterial sensors. In the context of pathogen detection, increased surface area can enhance the density and accessibility of imprinted cavities, facilitating more efficient interaction between the polymer and surface-exposed bacterial epitopes. Conductive nanomaterials, particularly when integrated into electrode-based systems, improve electron-transfer kinetics and signal-to-noise ratios, thereby enabling lower limits of detection for bacterial proteins in complex matrices. In addition, nanostructured scaffolds can promote thin-film or surface-confined imprinting architectures, which are especially important for recognizing epitopes embedded within large bacterial surface proteins without steric hindrance. Beyond signal enhancement, nanomaterials may contribute to improving fabrication consistency in bacterial sensing devices. Uniform nanostructured substrates can provide more controlled polymer growth, potentially reducing variability in film thickness, cavity distribution, and template extraction efficiency. Such structural control is critical for achieving reproducible bacterial recognition across sensor batches. While considerations of biocompatibility are relevant when sensors are intended for clinical sample analysis, the primary advantage of hybrid EIP–nanomaterial systems in this context lies in their ability to improve analytical sensitivity, robustness, and operational stability for rapid and selective bacterial detection.

7.3. Enhanced Biodegradability and Sustainable Sensor Materials

As EIPs move toward broader deployment in bacterial detection, particularly in disposable POC devices, material sustainability and safe disposal are becoming increasingly important considerations. The development of biobased and biodegradable monomers derived from renewable resources (e.g., lactic acid, glycolic acid, or polylactic acid derivatives) offers a more sustainable alternative to conventional methacrylate-based polymers commonly used in imprinting. For bacterial sensing applications, the primary motivation for enhanced biodegradability is not long-term implantation, but rather environmental impact and safe postuse disposal of single-use diagnostic devices. Sensors intended for large-scale screening of pathogens in clinical or food samples may generate substantial polymer waste. Designing EIPs from materials that degrade into nontoxic byproducts could reduce environmental burden while maintaining analytical performance. In addition, the application of green chemistry principles in EIP synthesis, such as reducing residual monomers, minimizing hazardous solvents, and optimizing energy-efficient polymerization methods, can improve the safety profile of sensors used in hospital or field settings. Importantly, any biodegradable or biobased polymer formulation must preserve the structural fidelity of imprinted cavities and maintain stable bacterial recognition under realistic assay conditions, including exposure to complex biological matrices. Future research should therefore focus on balancing environmental sustainability with analytical robustness, ensuring that biodegradable EIP materials retain sufficient mechanical stability, binding selectivity, and operational lifetime for reliable bacterial detection before controlled degradation occurs.

7.4. Multiepitope Imprinting

To address the growing complexity of bacterial infections and the rapid evolution of pathogenic strains, future EIPs are likely to advance toward multiepitope imprinting strategies specifically tailored for bacterial detection. Rather than imprinting a single peptide sequence, this approach involves incorporating two or more epitopes derived from the same bacterial protein, different virulence factors of one pathogen, or even distinct bacterial species relevant to a particular diagnostic context. In bacterial sensing, multiepitope imprinting offers several important advantages. First, simultaneous recognition of multiple surface-exposed regions enhances binding robustness, particularly when individual epitopes are partially masked or conformationally variable, while cooperative interactions between spatially related epitopes improve effective affinity and reduce false negatives in complex clinical matrices. Second, targeting conserved epitopes alongside strain-specific sequences balances universality and specificity, enabling broad detection across clinically relevant strains while discriminating closely related pathogenic and nonpathogenic speciesa feature especially valuable for rapid identification of antimicrobial-resistant strains. Third, multiepitope systems can improve reliability in mixed or polymicrobial infections, allowing for multiplexed or broad-spectrum bacterial sensing on a single platform without reliance on multiple biological receptors. The improved performance of dual-epitope imprinting arises from cooperative, spatially complementary binding sites that facilitate recognition of the intact protein or whole bacterium while reducing vulnerability to epitope masking. Generalizable design principles include selection of structurally stable, accessible epitopes, spatial arrangement to preserve cooperative interactions, and multimonomer polymer matrices that support multipoint noncovalent interactions, electrostatic, hydrophobic, and π–π, while minimizing nonspecific adsorption. Although this increased template complexity introduces challenges in cavity formation, cross-reactivity, and fabrication control, careful design and polymer optimization can maintain selective bacterial recognition. With these strategies, multiepitope imprinting represents a robust and adaptable pathway toward high-performance, clinically relevant EIP-based bacterial diagnostic systems.

7.5. Advanced Surface Modification

For EIPs intended for bacterial detection in clinical and food matrices, antifouling surface engineering is essential because complex samples such as blood, serum, urine, and milk contain abundant proteins, lipids, polysaccharides, and salts that can adsorb onto sensor surfaces, block imprinted cavities, and distort analytical signals. , Antifouling strategies can be grouped into three main classes. First, zwitterionic materials that suppress nonspecific adsorption primarily by forming a dense, charge-balanced hydration layer that creates an energetic barrier against protein and cell attachment. Second, polymer-brush architectures, including polyethylene glycol-based and zwitterionic brushes, reduce fouling through steric repulsion together with high interfacial water content; these features make them particularly useful when grafted onto the nonimprinted background surrounding the recognition sites rather than over the cavities themselves. Third, peptide-based and biomimetic antifouling layers provide compact hydrophilic and charge-balanced interfaces that resist nonspecific adsorption while remaining compatible with surface functionalization. In EIP design, these antifouling concepts can be integrated by copolymerizing zwitterionic monomers into the imprinting matrix, grafting antifouling brushes after cavity formation, or introducing localized peptide or hydrophilic interlayers that protect the surrounding surface without sealing the imprinted sites. , However, antifouling modification must be carefully optimized, because many surface coatings are not equally compatible with sensing, and the addition of biofunctional or protective layers can change the fouling behavior of the interface. Therefore, variables such as coating thickness, composition, and placement should be balanced to reduce nonspecific adsorption without compromising analyte access or analytical response. In addition to these antifouling approaches, biopolymer-based surface modification, particularly with chitosan or related hydrophilic matrices, can further improve film integrity, mechanical stability, and nanomaterial dispersion in hybrid EIP architectures, while also promoting more controlled polymer growth during surface-confined imprinting. , However, such materials may introduce extra functional groups that create additional electrostatic or hydrogen-bonding interactions with nontarget species, which can increase background adsorption and partially interfere with cavity accessibility in complex media. Therefore, surface modification must be carefully optimized to ensure that structural stabilization does not come at the expense of selective recognition.

7.6. Strategies to Mitigate Template Orientation in EIPs

To address the limitation of uncontrolled template orientation in conventional epitope imprinting, recent research has increasingly focused on strategies that impose spatial control on the epitope prior to polymerization. A prominent approach is surface or solid-phase imprinting, where the epitope is immobilized on a support to restrict its orientation and reduce template mobility during polymer growth. Immobilization can be achieved through covalent bonding, affinity interactions (e.g., boronate affinity), metal coordination, or self-assembled monolayers, enabling a consistent presentation of functional groups to the surrounding monomers and promoting the formation of uniform, accessible binding cavities. For instance, epitope tags or specific residues (e.g., cysteine, histidine) can be exploited for site-directed attachment to functionalized surfaces, yielding homogeneous orientation at the polymer interface that enhances both selectivity and binding kinetics. In controlled surface imprinting, polymers grow around these immobilized templates, resulting in imprinted cavities that are more uniform and exposed at the surface, which facilitates template removal and improves rebinding of the target protein. Additionally, techniques such as boronate affinity-anchored epitope imprinting have shown that immobilizing glycated peptide templates on functional substrates yields high-affinity and selective recognition of target proteins, demonstrating the value of orientation control in epitope imprinting design. Beyond chemical immobilization, hybrid imprinting techniques combining surface imprinting with advanced polymerization methods (e.g., atom transfer radical polymerization) or template-tag mediated assembly (such as histidine tags or aptamers) offer avenues to finely tune binding site orientation and accessibility. Collectively, these oriented imprinting strategies represent promising directions to overcome intrinsic EIP limitations and push the field toward reproducibly high-performance synthetic receptors for protein targets.

7.7. Portable and POC Diagnostic System

The future translation of EIPs for bacterial detection will be strongly influenced by the development of portable, miniaturized, and POC diagnostic platforms. Rapid identification of bacterial pathogens at the site of care, such as emergency departments, outpatient clinics, food production facilities, or environmental monitoring stations, can significantly reduce diagnostic delays and improve infection control measures. Integrating EIPs into lab-on-a-chip devices or microfluidic platforms offers the possibility of automated sample handling, reduced reagent consumption, and rapid detection of bacterial surface proteins without extensive laboratory infrastructure. For bacterial sensing applications, microfluidic confinement can improve mass transport of bacterial targets toward imprinted cavities, enhancing binding kinetics and shortening assay time. Surface-confined EIP films integrated with electrochemical, piezoelectric, or optical transducers are particularly well suited for compact device architectures, enabling real-time detection of specific bacterial epitopes directly in minimally processed samples. Smartphone-coupled readout systems represent another promising direction. By combining EIP-based electrochemical or optical detection with portable electronics, it may become feasible to perform decentralized screening of bacterial infections and transmit results for remote interpretation. Such platforms could support rapid triage decisions, antimicrobial stewardship efforts, and outbreak monitoring, particularly in low-resource settings where access to PCR or centralized laboratory diagnostics is limited. To achieve reliable field deployment, however, portable EIP-based bacterial sensors must demonstrate robustness against temperature variation, mechanical stress, and matrix interference, while maintaining stable and reproducible epitope recognition. Continued integration of microfabrication, antifouling surface engineering, and standardized fabrication protocols will therefore be essential for translating EIP technology from laboratory demonstrations to practical POC bacterial diagnostic systems.

8. Conclusion

EIP is emerging as a powerful strategy in bacterial biosensing, combining the molecular selectivity of biological recognition with the robustness and simplicity of synthetic polymers. By using short, surface-exposed peptide motifs instead of whole cells or full proteins, EIPs enable better-defined recognition cavities, improved site accessibility, and enhanced stability under harsh conditions. These advantages directly address key limitations of traditional bacterial- and protein-imprinted polymers, particularly template entrapment, structural heterogeneity, and poor reproducibility in complex matrices. Nevertheless, several challenges must be overcome before EIP-based sensors achieve widespread practical adoption. Rational epitope selection remains critical to ensure surface accessibility and specificity in real samples, while issues related to template removal, nonspecific matrix interference, and fabrication reproducibility continue to affect analytical reliability. Ongoing advances, including AI-assisted epitope design, nanomaterial-integrated architectures, and surface-confined imprinting strategies, are actively mitigating these limitations by improving binding fidelity, signal transduction, and device robustness. Overall, the continued convergence of rational molecular design, advanced polymer engineering, and portable sensing platforms positions EIP as a promising pathway toward next-generation bacterial biosensors. With further optimization and standardization, EIP-based systems have strong potential for translation into practical tools for clinical diagnostics, food safety, and environmental monitoring.

Ahmed S. El-tahlawy: Writingoriginal draft, Writingreview and editing, Formal analysis, Data curation. Mohamed Aly Saad Aly: Writingreview and editing, Methodology, Conceptualization. Stefano Cinti: Writingreview and editing, Methodology, Conceptualization. Aziz Amine: Writingreview and editing, Methodology, Conceptualization. Waleed Alahmad: Writingreview and editing, Supervision, Project administration, Methodology, Conceptualization.

The authors declare no competing financial interest.