Anti‐Fouling Polymer or Peptide‐Modified Electrochemical Biosensors for Improved Biosensing in Complex Media

Electrochemical biosensing represents a highly effective technology for detecting disease biomarkers given its high sensitivity, low and clinically relevant limit of detection, and cost effectiveness. However, in complex media such as urine, blood, sweat or saliva, biosensing performance can be significantly impacted by electrode biofouling by proteins, cells, lipids, and other matrix components. Such biofouling leads to reduced signal from the target analyte coupled with an elevated background signal, resulting in poor signal‐to‐noise ratios (SNRs), reduced sensitivity, and lower specificity. This comprehensive review describes the design of anti‐fouling polymers and peptides as a potential solution to prevent or suppress electrochemical biosensor fouling. Various anti‐fouling polymers and peptides developed for improved biosensing in complex media are summarized in the context of their mechanism(s) of anti‐fouling, methods of deposition, and practical applications. Recent advances and persistent challenges in the field are also reviewed to provide perspectives on new directions toward enhancing anti‐fouling in electrochemical biosensors.

or mercaptoundecanoic acid as backfiller in tandem with thiolated capture probes as biorecognition elements for reducing the interaction of biofoulants with electrode surface. [7]11][12] However, an MCH-based system can lead to lesser stability and non-reproducible results due to surface defects and heterogeneity of the coating/backfilling as well as displacement of the capture probe over time, leading to reduced storage stability; furthermore, the thiol-based interaction chemistry is exclusive to modifying gold electrode surfaces. [7,13][16] However, BSA attaches nonuniformly, desorbs gradually with time (including in response to the presence of higher affinity proteins often encountered in physiological fluids), and can hinder the electron flow from/to the electrode surface. [1,17]Furthermore, BSA is susceptible to aggregation at pH levels higher than its isoelectric point, which can present further stability challenges. [18]As such, neither of these solutions meets the ideal criteria of anti-fouling coatings being biocompatible, inert, maintaining the conductivity of the biosensor, having high anti-fouling properties, and being easy and fast to integrate within existing electrode manufacturing strategies.
In this review, we discuss the design and practical applications of anti-fouling polymers and peptides applied to address challenges in electrochemical biosensing targeting a wide variety of analytes and disease biomarkers.We detailed the mechanisms behind polymers/peptides commonly used for antifouling, methods for attaching them to biosensing substrates, and examples of the practical implementation of those strategies for biosensor design.We also identify emerging anti-fouling polymers and peptides with future potential in the context of electrochemical biosensing and ongoing challenges in this area to achieve better signal-to-noise ratios, better sensitivity, lower limits of detection (LOD), faster detection, higher specificity, improved ease of fabrication, and sustained stability.

Fundamentals of Anti-fouling in Electrochemical Biosensors
Figure 1 summarizes the mechanisms of anti-fouling, the main strategies for surface modification using polymers or peptidebased strategies, and standard methods for depositing such antifouling materials on electrochemical biosensors.Each of these aspects will be discussed in more detail in the subsequent sections.

Mechanisms of Anti-Fouling
Prevention of non-specific adsorption or anti-fouling of the surface typically involves one of three major mechanisms: the formation of a hydration layer, [8] steric repulsion, or electrostatic repulsion. [1,19]The formation of hydration layers, the most common approach, involves the attachment of polymers or peptides with a high capacity to bind water to the sensor interface, creating a hydration layer of water molecules at the interface that acts as a physical and energetic barrier that foulants must cross to attach to the surface. [1,2,8,20]Steric repulsion, in contrast, leverages the flexibility of macromolecular chains attached to an interface to resist fouling.The entry of a foulant into a brush-like steric repulsion layer results in a reduction of available volume for the chain and thus the effective compression of the chain, which for a flexible high-entropy polymer is particularly energetically unfavorable; the resulting chain expansion response aids in effectively repelling the foulants from the surface and preventing their deposition. [1,2,8,20]Electrostatic repulsion leverages the use of charged surfaces to minimize electrostatic attraction between the surfaces and charged foulants. [1,2,8,20]While this approach can be effective in more homogeneous fluid environments (including many environmental applications in which the vast majority of foulants are anionic), charge alone is less effective in a biological context given that many major foulants (e.g., proteins or lipids) can have both cationic and anionic charged domains, such that repelling one domain effectively attracts the other.

Types of Anti-Fouling Polymers and Peptides
Based on these three main mechanisms, three major classes of anti-fouling polymers or peptides have emerged that can promote anti-fouling through one or more of the mechanisms described. [21]

Poly(ethylene glycol) and Derivatives
Poly(ethylene glycol) (PEG)-based polymers are the current goldstandard for anti-fouling polymers. [22]The major mechanism of anti-fouling of such polymers is the formation of a hydration layer via the strong hydrogen bonding between the ether repeat units of PEG and water molecules, with each ethylene glycol in the PEG backbone able to strongly bind to one water molecule as determined through nuclear magnetic resonance (NMR) relaxation time measurements. [23]This result has also been confirmed via molecular simulation studies, in which an oligo(ethylene glycol) (OEG) self-assembled monolayer (SAM) demonstrated superior resistance to protein adsorption compared to a hydrophilic hydroxyl-terminated SAM. [24]However, the high flexibility of the ether bond in the PEG backbone also contributes to effective antifouling via a steric hindrance mechanism, with tuning of the surface packing density and chain length of PEG-based polymers enabling optimization of the anti-fouling properties through the combination of the two mechanisms.
Copolymers that incorporate PEG but can also hydrophobically interact with an underlying substrate have also demonstrated effective protein anti-fouling properties.Gao et al.  showed the effective anti-fouling properties of poly(methyl methacrylate(MMA)-co-methoxy poly(ethylene glycol) methacrylate (MPEGMA)) random copolymers following adsorption of the PMMA on polyethylene terephthalate or polyester, with sum frequency generation (SFG) vibrational spectroscopy and quartz crystal microbalance with dissipation monitoring (QCM-D) indicating that the disordered PEG units in the copolymer facilitated the formation of ordered interfacial water molecules via strong hydrogen bonding to provide resistance to protein fouling. [25,26]Linear PEG can be prone to oxidation and thus can be unstable in the electrochemical environment, [8] leading to increasing interest and emphasis around the engineering PEG-based materials with similar anti-fouling mechanisms but improved stability.Poly(oligo (ethylene glycol) methyl ether methacrylate) (POEGMA), a brush-like derivative of PEG, is also attracting attention given its capacity to be free radical (co)-polymerized and thus facilely incorporated into a range of different antifouling polymers. [27]Tuning the brush length of the OEGMA side chains to the methacrylate backbone can directly adjust the steric crowding of the PEG brushes and thus the effectiveness and mechanism of anti-fouling observed; [28] furthermore, the side chains can be converted to functional groups for biomolecular attachment if desired. [29]Du et al. demonstrated the higher stability of the POEGMA brushes in oxidative environments using molecular simulations and in situ QCM monitoring (addressing a key challenge with linear PEG materials), [30] while Hucknall et al. showed effective protein and cell resistance of POEGMA brushes grown on gold/glass using surfaceinitiated polymerization that enabled ≈100x lower LOD for an IL-6 protein microarray compared to a non-coated nitrocellulose membrane. [31]olyglycerol, a naturally-derived flexible polyether analogue to PEG, has also been demonstrated as an effective anti-fouling polymer following the same basic mechanism as PEG, with its high degree of branching providing particular benefits relative to linear PEG in terms of coating stability. [22,32,33]Siegers et al. demonstrated that polyglycerol-based SAMs on semi-transparent gold film showed comparable anti-fouling properties but better stability against heat and oxidation compared to PEG-based SAMs, [32] while Paez et al. observed lower fouling of surfaces coated with up to 9% amine-functionalized polyglycerol, although higher fouling was observed in polyglycerol coatings containing higher amine contents. [33]

Zwitterionic Polymers
Zwitterionic polymers are net electrostatically neutral polymers that have an equal number of positive and negative charges, typically in close proximity (≈2-3 carbons apart).The most commonly used zwitterionic polymers contain phosphorylcholine (PC), carboxybetaine (CB) or sulfobetaine (SB) units that consist of one anionic functional group (phosphate, carboxylate, or sulfate, respectively) and one quaternary ammonium group in each monomer sub-unit spaced by typically a small number (1-4) of carbon spacers.Compared to PEG, zwitterionic polymer coatings offer many experimentally and computationally demonstrated benefits.In particular, zwitterionic materials can more strongly bind water molecules due to both hydrogen bonding interactions as well as ionic solvation mechanisms, expanding their capacity for water structuring and thus their capacity to form dense hydration layers at interfaces. [2,34] recent computational study conducted by Liu et al. showed that the anti-fouling property of polymer brushes decreased in the order of PCBMA (poly(carboxybetaine methacrylate)) > PMPC (poly(2-methacryloyloxyethyl phosphorylcholine) > PS-BMA (poly(sulfobetaine methacrylate)) > PEG, consistent with the superior surface hydration enabled by zwitterionic polymers over PEG.[35] In addition, experimental studies involving 1 H NMR, atomic force microscopy (AFM), and fluorescence spectroscopy have noted that oligo(ethylene glycol) units exhibited weak hydrophobic interactions with proteins whereas PSBMA demonstrated no such interactions; [36] this result is consistent with other computational studies that have found that zwitterions have the least influence on protein structure and no observed alternations in hydrophobic interactions compared to PEG. [37] Moreover, zwitterions offer lower hydration free energy, indicating a stronger affinity for water molecules and a wider dipole orientation relative to PEG that makes it more challenging for potential foulants to displace water.[37] In addition to their inherent water binding capacity, zwitterionic polymers typically exhibit anti-polyelectrolyte behavior in that they can expand when exposed to higher salt concentrations that can screen the electrostatic interactions between the anionic and cationic functional groups in each monomer unit.For biosensor applications monitoring physiological fluids that typically have high salt contents, the capacity to hydrate upon salt exposure rather than collapse (as is typically the case with poly-mers) offers a key functional benefit relevant to anti-fouling.[38] Different zwitterionic polymers show varying degrees of swelling in response to changes in salt concentration, chain spacer length, and the concentrations and orientations of different types of anions.For example, neutron reflectivity analysis of PMPC indicated that it does not swell or deswell in response to changes in salt concentration or ionic strength, although the presence of ions can change the balance of intra/intermolecular forces and its stiffness; [39] in contrast, poly[3-(N-2-methacryloyloxyethyl-N,Ndimethyl)] ammonatopropanesulfonate (PMAPS) swelled significantly as the salt concentration increased.[40] The presence of chaotropic (weakly hydrated) or kosmotropic (strongly hydrated) anions can also compete with the zwitterionic polymer for bound water to either promote or deteriorate the anti-fouling properties due to changes in the hydration state and/or alterations in the strength of intramolecular electrostatic interactions, depending on the type of zwitterion and space between the charged groups present.[37,[41][42][43][44][45][46] Interestingly, a computational study by Shao et al. showed that carboxybetaine groups are more sensitive to kosmotropic cations while sulfobetaine groups are more sensitive to chaotropic cations, reflective of differences in the charge densities of the two zwitterionic groups.[37] The distance between the anionic and cationic groups also strongly influences the swelling and water binding response of a zwitterionic polymer.For example, Higaki et al. showed that the swelling sensitivity to ionic strength increases with an increase in the chain spacer length in poly(sulfobetaine) brushes; [44,45] while Huang et al. noted that poly(trimethylamine-N-oxide) (PTMAO) brushes with no carbon units between the cationic and anionic groups of the zwitterion displayed extremely high resistance to salt effects due to its shorter dipole and strong hydration capacity.[47] Changing the order of dipoles within the zwitterionic polymers can further alter polymer responses to salt.For example, Brown et al. recently showed that reversing the dipole orientation from poly(sulfothetin styrene) to poly(sulfonium sulfonate styrene) led to higher stability against nucleophiles due to a reduction in inter-zwitterion interactions, which are shielded in the case of poly(sulfonium sulfonate styrene) by the n-butyl groups extending from the chains.[48]

Hydration Layer-Supporting Peptides
Peptides are chains of amino acids that, in the context of antifouling, are typically used to impart hydrophilicity to the surface to increase local water binding. [21]Commonly used hydrophilic amino acids including glutamic acid (E), lysine (K), aspartic acid (D), arginine (R), and histidine (H) can be attached alternately or randomly through the formation of amide bonds to form antifouling peptides.The peptides can be charged or neutral depending upon their amino acid sequence; however, in terms of antifouling, the least amount of fouling is typically observed using zwitterionic peptides consisting of mixtures of charged amino acids that result in an overall neutral charge. [49]For example, Ye et al. observed that a self-assembled monolayer of a zwitterionic peptide (CRERERE) on a gold surface showed significantly better anti-fouling in comparison to an amphiphilic, non-ionic peptide (CYSYSYS), as determined using contact angle and SPR studies; in parallel, molecular dynamics (MD) simulations estimated that 55 water molecules could be bound around CRERERE but only 35 water molecules could be bound around CYSYSYS. [50] The hydrophilicity of the amino acids used to fabricate peptides chosen strongly influences the degree of fouling observed.For example, Chelmowski et al. showed that the SAM formed by a hydrophilic peptide showed the least protein adsorption when tested with streptavidin, BSA and fibronectin using surface plasmon resonance (comparable to gold standard OEGbased SAM) while comparably high protein adsorption was observed for hydrophobic peptide-based SAM and CH 3 -terminated SAM. [51]Similarly, Li et al. observed that the incorporation of hydrophilic amino acids such as serine into a peptide resulted in reduced interactions with protein foulants and thus improved antifouling performance relative to when hydrophobic amino acids such as leucine were incorporated. [52]he sequence of the amino acids can strongly influence antifouling, a particular beneficial feature of peptide-based antifouling agents given the ease of controlling amino acid sequences in oligomers relative to using other types of polymerization strategies.Chen et al. observed that peptides based on both alternatively and randomly arranged E and K amino acids can impart anti-fouling properties comparable to PEG-surfaces. [53]However, Li et al. observed that peptides produced by alternating E and K were significantly more anti-fouling than peptides produced with blocks of E and K in the presence of divalent cations, a result attributed to the increased inter-chain electrostatic interactions observed within the K block relative to more dispersed K residues; no significant difference being was observed in the presence of monovalent ions. [54]Changing the sequence of the amino acids can also affect the conformation of peptides due to differences in the resulting charge distribution, which in turn affects the anti-fouling performance.For example, Li et al. showed via MD simulations that zwitterionic peptides with different arrangements of E and K ranging from block to alternating resulted in different anti-fouling properties based on the height of the peptide brushes and the preferred inter/intrachain structures determined by the charge distribution along the peptide chain.Blockcharged peptides prefer loop and head-to-tail structures upon surface attachment whereas alternating-charged peptide prefer extended and parallel structures with dense brush structures; consequently, alternately-charged peptides performed better in antifouling applications. [55]eptides can also be tuned to bind to specific targets via the use of biorecognition sequences, including specific types of polymers or other biomaterials relevant for surface functionalization to create a more stable anti-fouling layer than would be achieved by simple physicochemical interactions.For example, Date et al. used a polyetherimide-specific polymer-binding peptide to observe the effect of peptide density on the further attachment of functional proteins to the surface.Streptavidin binding was saturated at 11% peptide density, above which amount of bound streptavidin started decreasing indicative of the clustering of peptides.The bound streptavidin was further used to attach capture DNA and hybridize it with complementary DNA to fabricate a biosensor platform. [56]verall, these three leading anti-fouling polymers/peptides offer both various benefits and challenges.PEG-based polymers remain considered as the gold standard for anti-fouling due to their hydration layer and steric hinderance anti-fouling mecha-nisms but are both insulative and susceptible to oxidation, potentially making them unstable in electrochemical environments in addition to resulting low electrochemical signals.PEG-based derivatives such as POEGMA, polyglycerol, etc. are more stable and less prone to oxidation but remain restricted by their insulative properties.Zwitterionic polymers are beneficial due to their ability to form stronger hydration layers, lower susceptibility to oxidation relative to PEG, and capacity for ion conduction but are much more easily deformed in electric fields due to their highly charged nature which may temper their benefits for some types of electrochemical sensing.Peptides provide high specificity and selectivity, are easily tunable, and can be easily integrated with biorecognition elements or on biosensing surface but are much more prone to enzymatic degradation and are both more expensive and more difficult to scale-up.As such, different materials may offer more significant advantages (and less significant disadvantages) depending on the application and electrical properties required for sensing.

Strategies for Forming Anti-Fouling Polymer/Peptide Coatings
Given the different synthetic routes available for preparing PEG (a step-growth polymer), zwitterionic polymers (most of which are chain growth polymers), and peptides (a condensation-based step growth polymer), various strategies need to be available to effectively tether, deposit, or otherwise irreversibly coat each type of polymer to the surface of an electrochemical biosensor such that stable anti-fouling performance can be achieved.

Self-Assembly
Self-assembly is one of the most common methods for preparing anti-fouling coatings, with the formation of SAMs of typically small molecule alkanethiols to gold surfaces being the most widely reported anti-fouling coating strategy.Zwitterionic end-terminated alkane thiol SAMs have attracted particular recent interest based on their improved anti-fouling properties, with a sulfobetaine-terminated SAM reported as an anti-fouling agent for the detection of prostate-specific antigen (PSA) using PSA-specific aminated aptamers [57] and a 2-methacryloyloxyethyl phosphorylcholine (MPC)-terminated SAM used successfully for in vivo electrochemical biosensing. [58,59]Wang et al. synthesized sulfobetaine-and carboxybetaine-terminated alkane thiols and found that SB-thiol showed improved anti-fouling performance compared to bare gold and CB-thiol, as determined by contact angle studies and SPR sensogram measurements with and without BSA (Figure 2).Cyclic voltammetry (CV) scans of both SAMs showed that SB-thiol forms a highly packed and thick layer while CB-thiol showed reduced obstruction to electron transfer and thus poorer anti-fouling. [60]Self-assembly in this context can facilitate the controlled orientation of molecules, precise control over film thickness, and the formation of high-density coatings; however, the stability of the coating (particularly in the presence of proteins that may provide competing gold-thiol interaction potential) can be marginal depending on the type of surface being coated. [61]Furthermore, since small molecule coatings are the Figure 2. A) Schematic of self-assembly of SB-and CB-thiol on gold surface, B) Amount of protein (BSA) adsorption on SB-thiol coated gold surface treated with different mole fractions of CB-thiol.Reproduced (Adapted) with permission. [60]Copyright 2019, American Chemical Society.
most commonly applied, the potential for leveraging steric repulsion for anti-fouling with traditional SAMs is minimal.
Polymers functionalized with thiol groups can be directly immobilized on gold substrates using the same interaction(s), thus introducing potential steric repulsion benefits to support antifouling relative to conventional small molecule-based SAMs.However, the structure of such coatings is typically much less ordered than a SAM based on the rotational energetic barriers inherent with reconfiguring long-chain polymers into ordered arrays.For example, PEGylated platinum bio-electrodes were reported by Yue et al. in which a thin thiolated PEG coating was self-assembled on platinum (Pt) electrodes, showing negligible impedance changes after fibrinogen incubation indicative of effective anti-fouling. [62]Correspondingly, Doneux et al. reported that a thiolated OEG 7 -SAM-coated electrode showed effective anti-fouling properties, although the nature of the redox test performed significantly affected the results.[65] As such, careful selection of the electrolyte solution is required to accurately assess antifouling properties of salt-sensitive polymers.
While this direct self-assembly based immobilization technique is one of the easiest ways to deposit polymer on a substrate, it can lead to relatively low grafting densities; this can represent a particular challenge in the context of PEG-based materials in which graft density is an essential determinant of anti-fouling function.SAMs require time to self-assemble, reducing the potential throughput and scalability of the technique for large-scale biosensor fabrication.Furthermore, the limited number of surfaces directly amenable to such modification (i.e., gold or potentially platinum [66] ) as well as the limited availability and long-term storage stability of polymers functionalized with thiol groups represents a barrier toward broad utilization of this approach. [61]

Chemical Grafting
As an alternative to gold-thiol self-assembly, polymers can also be covalently anchored on surfaces using grafting strategies.Two strategies are typically used for grafting: 1) graft-to, in which a pre-fabricated anti-fouling polymer end-functionalized with a specific functional group is tethered to an electrode surface; or 2) graft-from, in which a polymer is grown from an electrode surface (typically from a pre-tethered surface initiator).For either type of grafting, traditional SAMs are often leveraged to introduce to the surface either a conjugation site for graft-to strategies or an initiator for graft-from strategies. [61]However, such an approach still limits grafting to a gold electrode surface and requires specifically end-functionalized polymers to facilitate grafting to the functional SAMs prepared.
As an alternative strategy to allow for graft-to surface grafting without the need for specific surface chemistries and/or polymer functionalization, polydopamine (PDA)-based coatings have recently attracted extensive attention.PDA-based coatings form due to the oxidative self-polymerization of dopamine in alkaline conditions. [67,68][69] The diversity of adhesive interactions thus facilitated by PDA makes it highly amenable for facilitating graft-to modification of a variety of surfaces using a variety of polymers.For example, Pop-Georgievski et al. reported grafting of poly(ethylene oxide) to a self-polymerized dopamine-coated surface, [67] while Asha et al. reported grafting of an anti-fouling dopamine/phosphorylcholine copolymer on a polyethylenimine (PEI)/polydopamine (PDA) co-deposited surface that also facilitates amine functionalization of a variety of surfaces. [70]However, it should be noted that any graft-to process (regardless of how the polymer attaches to the surface) typically leads to lower graft densities based on the tendency of the polymer to graft in its random coil rather than extended form as well as a less uniform distribution of anti-fouling polymers/peptides relative to other techniques. [61]he graft-from method enables the formation of higher density and more extended grafts but also requires much more complex fabrication steps.Most commonly, a SAM-based polymerization initiator is self-assembled on the electrode surface and used to seed a controlled radical polymerization reaction such as atom transfer radical polymerization (ATRP), which requires strict air-free conditions and precisely balanced catalysts to properly function. [61,71]Various anti-fouling coatings have been reported based on surface-initiated ATRP (SI-ATRP) from an initially bound SAM-initiator.Chen et al. reported a SI-ATRP process for grafting the zwitterionic polymer poly(3-(1-(4-vinylbenzyl)−1Himidazol-3-ium-3-yl) propane-1-sulfonate) on a gold electrode from the SAM initiator -mercaptoundecyl bromoisobutyrate, [72] Santos Pereira et al. formed polymer blocks of POEGMA and carboxybetaine acrylamide on gold using the SAM initiator mercaptoundecyl bromoisobutyrate, [73] Riedel et al. reported a SI-ATRP process for grafting poly(hydroxy-capped oligoethylene glycol methacrylate) (poly(HOEGMA)) and POEGMA brushes on gold using a similar initiator, [74] and Parrillo et al. reported the SI-ATRP of POEGMA as bottom polymer block and glycidyl methacrylate as top functionalizable polymer block using the same SAM initiator pre-assembled on a gold surface. [75]I-ATRP has also been utilized to graft-from non-gold surfaces following attachment of the initiator to the surface using other adhesion techniques.For example, Kuang et al. reported significantly reduced surface fouling using sulfobetaine methacrylate (SBMA) brushes fabricated using a bifunctional tripeptide bromide as initiator on metal, metal oxide and polymeric substrates, leveraging the adhesion properties of the L-3,4-dihydroxyphenylalanine (DOPA) amino acid used to fabricate the peptide. [76]Conversely, Ryu et al. adhered a catecholaminebased peptidomimetic anti-fouling polymer (PMAP) to a TiO 2deposited quartz surface using a graft-to approach, observing 2-10 fold better anti-fouling properties than methoxy PEG (mPEG) quartz surfaces. [77]o address the drawbacks of more traditional ATRP techniques such as its air sensitivity and the requirement for a high concentration of toxic transition metal catalyst, newer ATRP techniques have attracted interest for preparing anti-fouling surfaces while mitigating various limitations of normal ATRP.As a particular example, activators regenerated by electron transfer (ARGET)-ATRP represents a limited air-tolerant strategy that uses a lower amount of Cu (II) and a higher amount of environmentallyfriendly reducing agent to regenerate the activator without producing initiators that would initiate new chains. [78]For example, Kang et al. used SI-ARGET ATRP to graft poly(CB) under air-open conditions using a SAM initiator on a gold surface and achieved low fouling against fibrinogen. [79]In comparison with SI-ATRP, ARGET-ATRP is more cost-effective and environmentally-friendly while also enabling the fabrication of thicker polymeric films with higher grafting efficiency under ambient conditions. [80]

Photopolymerization
Photopolymerized coatings in which the polymerization of chain-growth monomers can be surface initiated using radiation (typically UV or gamma) have also attracted increasing interest.Photopolymerization is fast (a particular advantage compared to SAM-based approaches) and is particularly useful for preparing thin film hydrogels on electrode surfaces, including thin film composite hydrogels that can include conductive components to help circumvent the insulative properties of SAM and (even more acutely) polymer-based coatings.For example, Zinggeler et al. described the preparation of screen-printed electrodes (SPEs) incorporating a UV-cross-linked thin hydrogel film composed of conductive carbon nanotubes (CNTs) and the anti-fouling copolymer poly(N,N-dimethylacrylamide-statmethacryloyloxy-benzophenone) (Figure 3A).The 2.5/1 ratio of polymer/CNTs was identified as the most effective combination due to its highest electroactive surface area (EASA) and highest anti-fouling properties against BSA, with only a very small decrease in EASA observed after 1 h (Figure 3B). [81]The prepared electrodes enabled the successful electrochemical detection of the inflammatory biomarker C-reactive protein (CRP) using chronoamperometry in undiluted human blood serum, using a CRP capture antibody (Ab) as the ligand.Similarly, Chocholova et al. reported the fabrication of thin film poly{4-[(3-methylacrylamidopropyl)dimethylaminobromobutyrate]-co-[N-methacryloyl-4-azidoaniline]} and poly{4-[(3-methylacrylamidopropyl)dimethylamino-bromoacetate]-co-[N-methacryloyl-4-azido-aniline]} (CBAmN 3 copolymer)-based hydrogels on carbon SPEs that were further functionalized with human epidermal growth factor receptor 2 (HER2) antibodies, facilitating the accurate detection of the breast cancer biomarker HER2 protein [82] Relative to photopolymerization, grafting via gamma ray initiation offers several advantages including the elimination of the need for catalysts or initiators and the ability to graft with proteins and other biomolecules at low temperatures.For example, Jeong et al. grafted MPC on polypyrrole (PPy) electrodeposited gold electrodes, with the negligible change observed in charge transfer resistance (R CT ) upon incubation in a BSA solution indicating high anti-fouling properties and electrical properties similar to PPy electrodes without MPC grafting. [83]However, gamma irradiation requires specialized instrumentation and safety protocols that can limit its utility for scalable commercial biosensor production.
Surface-initiated photo-iniferter-mediated polymerization (SI-PIMP), which utilizes SAMs of UV-cleavable photoiniferters, e.g., dithiocarbamate derivatives that can serve as an initiator, terminator, and chain transfer agent for polymerization, has also been reported as an effective method for preparing antifouling surfaces. [84,85]For example, Krause et al. reported the formation of an anti-fouling carboxybetaine polymer thin film formed using UV irradiation in the presence of carboxybetaine and a thiol-based dithiocarbamate iniferter self-assembled on a gold surface [86] while Liu et al. reported serine methacrylate grafting on an iniferter self-assembled gold surface using UV irradiation to form an anti-fouling amino acid-based zwitterionic homopolymer-coated surface; [87] both surfaces showed better anti-fouling properties than the corresponding unmodified surface.Using this iniferter/SI-PIMP approach has also been shown to allow for better control over the polymerization reaction and the thickness and uniformity of polymer films relative to conventional photopolymerization, with film thickness directly controllable based on the photoirradiation time. [87]Additionally, compared to SI-ATRP, SI-PIMP does not require toxic transition metal catalysts and allows for the polymerization of more polar monomers that complex with the transition metal catalyst and thus cannot be polymerized using ATRP. [87]Copyright 2022, American Chemical Society.

Electrografting
Electrografting via the electrochemical reduction of diazonium salts is another widely used method to fabricate anti-fouling polymer-coated surfaces for electrochemical biosensing.The process involves the reaction of aromatic amines with sodium nitrite and an acid to form a diazonium salt that is subsequently reduced electrochemically, yielding an aryl radical that can bind to the surface as well as facilitate reactions with other already surfacebound or solution-based molecules to induce surface dendrite growth.90] Electrografting has been reported to out-perform self-assembly in some contexts for the fabrication of electrochemical biosensors.For example, Gui et al. compared zwitterionic-phenyl and OEG-phenyl electrografted layers on glassy carbon electrodes (GCEs) with OEG alkanethiol SAMs on gold and observed that the electrografted layers exhibited lower impedance than OEG-SAMs while maintaining similar anti-fouling properties. [91]Jiang et al. reported electrografting of a phenyl phosphorylcholine (PPC) diazonium salt on indium tin oxide (ITO) electrodes to create an anti-fouling coating that showed high anti-fouling properties when tested against human serum albumin (HSA). [92]Similarly, Parviz et al. reported electrografted PPC on a gold surface and observed lower impedance but also poorer anti-fouling properties relative to dodecane thiols SAM on gold, attributed to the lower density and loose multilayer formation of the PPC layer and/or its failure to maintain a charge-neutral state; however, PPC with a lipoamide group demonstrated anti-fouling properties equivalent to those of OEG on gold (OEG/Au) due to its charge neutrality, higher density, and more compact molecular layer. [93]lectrografted coatings have also been successfully integrated with recognition ligands to enable biosensing in physiological fluids.For example, PPC and phenyl butyric acid (PBA)grafted ITO electrodes were used to detect tumor necrosis factor  (TNF-) in whole blood, with the PBA-derived surface functional groups used to attach both antibodies specific to TNF- and horseradish peroxidase (HRP)-labelled secondary antibodies used to generate an electrochemical signal if target binding oc-curs (Figure 4A).The prepared anti-fouling electrodes showed the least change in CV peak separation and peak current after HSA incubation (Figure 4B), while electrochemical impedance spectroscopy (EIS) analysis showed a ≈35% increase in R CT after HSA incubation. [94]hile electrografting offers several advantages over selfassembly (in particular, enhanced coating stability and enhanced fabrication speed), electrografting can result in disordered polymerization and the formation of thicker coatings on the electrode surface.This thicker layer can act as a barrier to electron transfer, increasing the electrode's impedance or charge transfer resistance as observed by Parviz et al. during the electrografting of PPC on gold. [93]A study by Taufik et al. reported a similar observation, wherein ethylene oxide SAMs formed directly on the surface demonstrated higher anti-fouling ability compared to OEG attached to the aryl diazonium layer on the electrode surface. [95]ndeed, in many cases, it is sought to minimize rather than promote the formation of polymer layers as opposed to monolayer formation on the surface, most commonly utilizing steric hindrance and/or radical scavenging strategies to limit dendrite formation. [96]Electrografting is also significantly limited by the very narrow range of molecules that can be successfully electrografted, the limited commercial availability of suitable molecules for electrografting, and the frequent requirement of additional molecules like PBA for facilitating attachment with biorecognition molecules, adding to the cost and complexity of the system.

Electropolymerization
Electrochemical polymerization or electropolymerization is a well-known method for polymer deposition on a conductive electrode surface, most commonly employing anodic electropolymerization of oxidizing monomer species.During this process, an oxidative monomer radical cation is generated due to the loss or transfer of an electron from the monomer to the electrode when an electric potential is applied.The formed radical cation can either combine with another radical cation to facilitate growth or react with a monomer to form a polymer chain firmly bound to the electrode surface. [97]The thickness of the polymer film formed from electropolymerization can be controlled by adjusting parameters such as the potential window and the polymerization time. [97]igure 4. A) Electrografted diazonium salts with zwitterionic (PPC) and carboxylic (PBA) functional groups on ITO electrodes as an anti-fouling electrochemical biosensor for the detection of TNF-.B) CV curves of the electrografted surface before and after incubation in HSA.Reproduced (Adapted) with permission. [94]Copyright 2016, American Chemical Society.
Electropolymerization of conductive polymers has been reported for electrochemical biosensing along with maintaining higher anti-fouling properties.For example, Zhao et al. electropolymerized polyaniline (PANI) and polythionine (PThi) in presence of a hydrophilic cross-linker on a GCE as an anti-fouling hydrogel coating for the electrochemical detection of cancer antigen 125 (CA125) in human serum.Co-deposition of PANI and PThi was performed using aniline, thionine and a highly hydrophilic cross-linking agent (phytic acid) by conducting 6 cycles of CV from −0.2 to 1 V followed by the electrodeposition of gold nanoparticles (AuNPs) at a constant potential (−0.2 V) for 30 s using chronoamperometry.The prepared coating showed a nearly unchanged square wave voltammetry (SWV) response after 12 h of incubation in 100% serum, while the concentration of CA125 detected in human serum was comparable to that obtained from ELISA, demonstrating the efficacy of the anti-fouling hydrogel for CA125 detection. [98]Similarly, Wang et al. reported a conductive supramolecular polymer hydrogel (CSPH) made of electropolymerized PANI and poly(m -aminobenzoic acid) (PABA) on a GCE for the electrochemical detection of thrombin.PANI-PABA was electropolymerized using chronopotentiometry at a constant current density of 0.01 mA cm −2 for 1 h on the electrode in the presence of aniline, 3-aminophenylboronic acid hydrochloride (APBA), and HClO 4 as the electrolyte solution; the boronic acid groups of the APBA residues were then further linked via boronate ester bonds with hydroxyl groups on polyvinyl alcohol (PVA).The differential pulse voltammetry (DPV) signal was reduced by only 6% after incubation in 100% fetal bovine serum (FBS) for 30 min, with effective thrombin detection achieved in human serum using a DPV-based electrochemical assay in which thrombin was sandwiched between a capture aptamer attached on CSPH and signal aptamer-coated magnetic particles. [99]lectropolymerization of conductive polymers containing side chains of zwitterionic moieties (in particular, zwitterionicfunctionalized poly(3,4-ethylenedioxythiophene) (PEDOT)) has also been used to create conductive anti-fouling coatings.Cao et al. reported the electropolymerization of sulfobetainefunctionalized 3,4-ethylenedioxythiophene (SBEDOT) monomer in an aqueous solution containing LiClO 4 electrolyte, which was electropolymerized on ITO-coated polyethylene terephthalate (PET) or gold electrodes by CV using voltage cycling from −0.6 to 1.3 V or by a galvanostatic method operating at 0.1 mA s −1 .
The galvanostatic method resulted in a more homogenous film than the CV method; however, the impedance characteristics of the prepared electrodes were comparable to that achieved with PEDOT, affirming the dense packing of the polymer on the gold electrodes that led to effective anti-fouling properties versus blood and serum. [100]Wu et al. used a similar PSBEDOT coating on a Pt electrode in the presence of glucose oxidase enzyme for glucose sensing, showing very high stability in human blood plasma. [101]Zhang et al. evaluated the anti-fouling properties of electropolymerized PEDOT containing OEG and zwitterionic side chains on gold electrodes; upon electrically stimulating the electrodes by applying 1000 CV scans from −0.3 to + 0.3 V, only electrodes coated with phosphorylcholine-functionalized PEDOT (PEDOT-PC) showed better electrochemical stability and antifouling properties versus proteins (e.g., BSA, fibrinogen) and cell adhesion compared to PEDOT electrodes, attributed to the balanced charge density of PC groups. [102]Liu et al. used a similar PEDOT-PC coating on carbon fiber microelectrodes for the in vivo electrochemical detection of dopamine, showing effective anti-fouling upon the incubation of the electrodes in 10 mg mL −1 BSA while successfully detecting dopamine in vivo in rat brains. [103]lectro-copolymerization can further be applied for tuning the conductive and anti-fouling properties of zwitterionic PEDOT-based polymers.For example, Goda et al. prepared electro-copolymerized coatings based on EDOT-co-EDOTPC (a phosphorylcholine-containing copolymer), EDOT-co-EDOTCB (a carboxybetaine-containing copolymer), and EDOT-co-EDOTSB (a sulfobetaine-containing copolymer) using CV from −0.6 to +1.4 V on a GCE (Figure 5A) and showed that all three zwitterionic EDOT monomers added at both 25 and 50 mol% comonomer content enabled better anti-fouling than homopolymer PEDOT (Figure 5B), although higher anti-fouling capacity was correlated with reduced surface conductivity. [104]nti-fouling zwitterions mimicking ionic liquids (ILs) such as [1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide] have also been electropolymerized and used as antifouling coatings for electrochemical biosensing.For example, Song et al. reported the electropolymerization of IL-doped PE-DOT on a GCE using CV from −0.5 to 1.2 V for 2 cycles.The neutral charge and hydrophilicity of the coating imparted superior anti-fouling properties, enabling the detection of dopamine Figure 5. A) Electropolymerization of EDOT with PC-or CB-or SB-functionalized EDOT on glassy carbon electrodes.B) Protein adsorption studies using QCM on glassy carbon electrode (GCE) surfaces electropolymerized with 0-50 mol% of EDOTPC, EDOTCB, EDOTSB and incubated in 100% human serum for 30 min.Reproduced (Adapted) with permission. [104]Copyright 2019, American Chemical Society.
in the presence of BSA, HSA, lysozyme, and human serum using CV. [105]s an alternative approach, electropolymerization has been reported as a method to graft initiator groups to an electrode surface to enable subsequent types of surface functionalization.For examples, Ashraf et al. electropolymerized a P(EDOT-co-EDOTBr) coating on an electrospun fiber mat and then used the Br residues in the EDOTBr comonomer as an initiator for SI-ATRP grafting of POEGMA brushes, resulting in significantly improved anti-fouling properties relative to surfaces with no POEGMA brush coating. [106]Another ATRP-related polymerization, surface initiated-activator generated by electron transfer (SI-AGET) ATRP, has been used in a similar manner but offers the advantage of using non-radical forming reducing agents such as ascorbic acid or tin 2-ethylhexanoate, avoiding the for-mation of byproducts that might create new initiated chains in solution while also reducing dissolved oxygen to allow polymerization to proceed in the presence of air. [107,108]Hackett et al. used this approach to graft anti-fouling (poly(ethylene glycol) methyl ether methacrylate (PEGMMA)-co-(diethylene glycol) methyl ether methacrylate (DEGMMA)) brushes from a similar P(EDOT-co-EDOTBr) coating electropolymerized on a gold electrode. [109]part from decoupled anodic/cathodic electropolymerization followed by normal ATRP, ATRP can be utilized directly in an electrochemical context for the preparation of anti-fouling coatings to enable electrochemical biosensing.Electrochemicallymediated atom transfer radical polymerization (eATRP) involves a three-electrode system in which electric potential is applied to reduce air-stable copper (II) deactivator into copper (I) activator that acts as a switch for polymerization.The monomer to be polymerized reacts with active Cu(I)-Br, forming dormant Cu(II)-Br 2 and free radicals that can induce polymer chain growth.Polymerization control is enabled based on the conversion between the active and dormant state depending on applied potential and polymerization time. [110]eATRP offers several advantages over other related strategies including precise control over the activator:deactivator ratio and polymerization rate, enhanced resistance to the presence of oxygen, elimination of the need for reducing agents, and the production of environmentallyfriendly coatings. [111]As an example of this approach, Hu et al. utilized eATRP technique to electropolymerize N-(3-sulfopropyl)-N-(methacryloxyethyl)-N, N-dimethylammonium betaine in the presence of tris(2-pyridylmethyl) amine (TPMA), CuCl 2 , and glucose using 20 cycles of CV between −1.0 and 0.6 V.The prepared electrodes detected glucose with negligible reduction in sensitivity following incubation in bovine serum for 15 days, demonstrating the anti-fouling properties of the coating. [112]imilar to conventional ATRP, eATRP can be localized at an electrode surface by pre-coating the surface with a suitable initiator molecule, a process referred to as electrochemically induced surface-initiated atomic-transfer radical polymerization (e-SIATRP). [113][115][116] As representative examples, e-SIATRP was used by Zhao et al. for preparing polymer brushes of sulfobetaine vinylimidazole (SBVI) on a 3-(trichlorosilyl)-propyl-2-bromo-2-methylpropanoate initiatormodified Si wafer (using silane chemistry) using CV in a 2:1 water/methanol solution containing SBVI, benzyltributylammonium chloride, CuCl 2 and CuII/bipyridine (bipy) complex.When a negative potential was applied on a working electrode kept near the initiator-modified surface, Cu(I) ions were generated from Cu(II), triggering the polymerization of the monomer present in the solution to form a coating with anti-fouling properties against BSA and lysozyme as confirmed using QCM. [114]Similarly, Hu et al. used e-SIATRP to fabricate PSBMA-based hydrogel coatings on gold-deposited silicon electrodes that showed delayed decay in the impedance-time scans by EIS indicative of anti-fouling behavior attributed to the presence of the PSBMA coating. [117]hile electropolymerization can enable precise control over coating thickness, fast fabrication, and facile processing, it also has drawbacks including the limited availability of anti-fouling compounds that can be electropolymerized, its often low graft yield, and the potentially poor solubility of the electropolymerized product. [97]One potential solution to expand the scope of anti-fouling materials available for electropolymerization is to codeposit non-conductive polymers with known anti-fouling properties with other electroactive monomers during the electropolymerization process.For example, while hyaluronic acid (HA) is a highly water binding polymer known for its anti-fouling properties, it is not electrochemically active; however, co-deposition of electroactive and bioadhesive dopamine (DA) with HA can en-able the fabrication of electrodes with significantly reduced contact angles and resistance to protein adsorption. [118]

Commonly Used Anti-Fouling Polymers/Peptides
In the following section, we describe strategies in which a combination of one of the three main classes of anti-fouling polymers or peptides, one or more assembly/deposition technique, and a biosensing ligand was used to create functional electrochemical biosensors with improved anti-fouling performance in real physiological fluids.Table 1 provides a comprehensive summary of reported biosensors that utilize one of the three key anti-fouling polymers/peptides (PEG, zwitterionic polymers, or peptides) previously discussed in this review.

PEG-Based Polymers
PEG coatings have been used directly to create functional electrochemical biosensors with reduced fouling.Wang et al. reported PEG coated EIS-based electrochemical biosensor for the effective detection of adenosine triphosphate (ATP) in complex media.To prepare the electrochemical biosensor, a glassy carbon electrode (GCE) was coated with polydopamine followed by the self-assembly of thiolated aptamer and thiolated PEG on polydopamine-coated GCE using a Michael addition reaction, after which the electrode was backfilled with MCH.The prepared aptasensor showed only a ≈5% change in R CT when incubated with 0.5 mg mL −1 protein (lysozyme, -lactoglobulin, BSA) in comparison to the 50%-70% change observed in the absence of PEG; furthermore, when incubated in human plasma, the PEG-coated electrode showed a ≈60% change in R CT in comparison to the 125% change observed in absence of PEG.The fabricated PEG-based electrodes enabled the detection of ATP with a LOD of 0.1 pM in buffer and a recovery distribution of 98.4-106.1% in 3% human plasma samples; the sensor could also detect ATP in centrifuged breast cancer cell lysates with results equivalent to those obtained from high-performance liquid chromatography (HPLC) (0.27 (±0.02) mM). [119]As another example, Wang et al. reported a PEG-grafted electrochemical biosensor for the detection of microRNA (miRNA) based on DNA/RNA hybridization, with electrochemical detection enabled by a methylene blue signal recorded using DPV.A glassy carbon electrode was coated first with PPy nanowires using electrochemical polymerization, after which the PPy nanowires were coated with 4armed PEG terminated with amine groups by electrochemically oxidizing the terminal amine groups on PEG to covalently immobilize them on PPy nanowires.The obtained electrode was further used for carbodiimide-mediated immobilization of carboxylfunctionalized and miRNA-specific DNA to the residual amine groups of PEG to facilitate ligand-specific biosensing (Figure 6A).A negligible change in R CT was observed when the prepared sensor was incubated for 30 min in up to 10% human serum, although increased resistance was noted at higher serum concentrations (Figure 6B).The prepared sensor detected miRNA with PEG/PPy nanowires on GCE [120] Covalent DNAzyme-immobilizing microgel magnetic beads [ 123] Semi-batch inverse suspension polymerization SWV E. coli using DNAzyme 1000x higher electrochemical signal using microgel magnetic beads compared to Dynabeads.10x reduction in signal when tested in urine compared to a 2000x reduction with Dynabeads (both for 60 min).
a LOD of 0.033 pM in buffer (Figure 6C), with the signal recoveries distribution ranging from 95.2% to 103.7% in 5% serum samples. [120]As such, while PEG-only coatings can improve antifouling while maintaining sensor performance, significant signal losses are still observed unless the physiological fluid is significantly diluted in most cases.
To compensate for the insulative properties of PEG, coatings have been formed in which PEG was used in combination with conductive polymers to enhance the signal-to-noise ratio of antifouling biosensors.For example, Hui et al. reported the use of PANI/PEG nanofibers for detecting the breast cancer gene 1 (BRCA1) biomarker in complex human serum samples by covalently immobilizing PEG on PANI nanofibers that were first electrodeposited on GCEs.The resulting coating showed both improved conductivity relative to PEG-only coatings and improved anti-fouling properties upon incubation in protein solutions and complex media as determined using DPV. [121]As another example, a coating consisting of graphene oxide functionalized with metalloid polymer hybrid nanoparticles consisting of silver metal, silica non-metal, and PEG on a gold-printed circuit board electrode enabled the direct electrochemical detection of quercetin with a LOD of 3.57 nM in buffer, [122] although sensing performance was not reported in real physiological samples.
To avoid potential challenges around the electrochemical stability and the emerging immunogenicity of PEG, PEG-derivative polymers including POEGMA, and polyglycerol have recently attracted more interest in electrochemical biosensing.For example, our group has reported anti-fouling microgel magnetic beads (mMB) based on POEGMA to immobilize an Escherichia coli (E.coli)-specific cleavable methylene blue-tagged electroactive DNAzyme.The electroactive strand cleaves from the DNAzyme upon interaction with E. coli to enable its detection on capture DNA-immobilized nanostructured gold electrodes using SWV measurements (Figure 7A).Use of the anti-fouling POEGMAbased magnetic beads resulted in higher current densities at 10 5 CFU mL −1 bacteria concentrations in both buffer and clinical human urine samples from urinary tract infection patients compared to commercial magnetic beads (cMB) (Figure 7B), enabling the detection of 6 CFU/mL and 138 CFU/mL of bacteria in buffer (Figure 7C) and undiluted urine, respectively (Figure 7D). [123]a et al. electropolymerized hyperbranched polyglycerol (HPG)functionalized EDOT (EDOT-HPG) monomers on a GCE and functionalized the obtained PEDOT-HPG coating with alphafetoprotein (AFP) antibody via Schiff base formation, taking advantage of the high density of reactive end groups on the highly branched HPG.The prepared platform detected AFP with a LOD of 0.035 pg mL −1 in buffer and reported a 6% difference in readings obtained in human serum compared to detection achieved with a conventional electrochemiluminescence (ECL) assay. [124]

Zwitterionic Polymers
Most reported examples of zwitterion electrochemical biosensor coatings in the literature combine a zwitterionic polymer with a conductive polymer/nanomaterial to address the insulating properties of a polymer electrode coating.Kilic et al. reported  [123] Copyright 2022, American Chemical Society.zwitterionic polypyrrole (ZiPPy) electropolymerized electrodes for the impedance-based detection of SARS-CoV-2 antibodies in human saliva.A zwitterionic pyrrole (ZiPy) monomer was synthesized and then electropolymerized on carbon and/or gold electrodes in presence of the spike protein of SARS-CoV-2 to prepare spike protein-and zwitterionic polypyrrole (ZiPPy)coated electrodes.Including a zwitterionic component in polypyrrole screened the cationic charge of PPy and thus significantly reduced protein adsorption.The prepared biosensor detected SARS-CoV-2 antibodies with a LOD of 50 ng mL −1 in both centrifuged and undiluted saliva (Figure 8). [125]Zhang et al. reported a biosensing platform consisting of a self-polymerized dopamine and silver nanoclusters (AgNCs) layer (the latter to improve conductivity) on a GCE on which a catechol and zwitterionicbifunctional PEG (b-PEG) polymer was attached via catechol chemistry and an ATP aptamer was attached to the AgNCs or PDA.The presence of b-PEG improved the anti-fouling properties of the surface to enable the detection of ATP in human plasma within 2 h with a LOD of 0.01 pM in buffer and recovered 93.7-106.1% of ATP in 1% human plasma/serum compared to ATP spiked in the samples. [126]Wang et al. reported the use of photopolymerized CBMA on electrodeposited PANI nanowires for the detection of carcinoembryonic antigen (CEA) in human serum with a low LOD of 3.05 fg mL −1 in buffer along with effective anti-fouling properties in complex media such as cow's milk, serum, fetal bovine serum (FBS) and saliva.The prepared biosensor detected CEA in 10% human serum and observed recovery of 94.3%-104.2%compared to CEA spiked into the same fluids. [127]elatively fewer examples of zwitterionic coatings without an accompanying conductive component have been reported as effective electrochemical biosensors.Monomethoxy-poly-(ethylene glycol)-b-poly(l-lactide)-b-poly(sulfobetaine methacry-late) (mPEG-PLA-PSBMA), [128] catechol-g-poly(2-(methacryloyloxy)ethylphosphorylcholine-p-nitrophenyloxycarbonyl-poly-(ethylene glycol) methacrylate), [129] and carboxybetaine polypeptoid hydrogels [130] have all been reported to exhibit excellent anti-fouling properties, but to our knowledge none have been used for electrochemical biosensing.However, Xu et al. reported successful electrochemical biosensing of self-copolymerized zwitterionic SBMA and dopamine on GCEs for the detection of CEA in human serum.Despite the poor conductivity of the coating, the prepared system detected CEA with a LOD of 3.3 fg mL −1 in buffer, showed good anti-fouling properties in clinical human serum samples, and could match ECL readings in serum. [131]As such, thin zwitterionic polymer-only coatings without conductive additives may be worth further research attention.
Given the relatively high charge density of zwitterionic polymers, it is possible that zwitterionic polymer structure may be altered by the electric field applied during biosensing; [20,132] if this were to induce deswelling in the zwitterionic layer, the antifouling properties may be negatively affected.While none of the studies reported directly investigated such an effect during electrochemical biosensing, further consideration on this point is likely warranted to improve the design of zwitterionic antifouling interfaces.

Peptides
While multiple hydrophilic peptides have been explored as antifouling coatings, zwitterionic peptides have been particularly exploited for use as anti-fouling coatings on electrochemical biosensors.For example, Wang et al. reported a macroporous gold-coated peptide and aptamer-based electrochemical interface Figure 8. A) Electropolymerization of electrodes with anti-fouling zwitterionic-polypyrrole (ZiPPy).B) Impedance change % of prepared ZiPPy electrodes after incubation in human serum, saliva and 10% BSA for 1 h to determine the anti-fouling characteristics.C) Prepared electrodes coated with spikeprotein of different variants used for detection of SARS-CoV-2 antibodies.D) Different antibody concentrations spiked in saliva were measured using the prepared ZiPPy electrodes, resulting in a linear resistance response to antibody concentration.Reproduced (Adapted) with permission. [81]Copyright 2022, John Wiley and Sons.
for detecting IgE in serum by sequentially self-assembling thiolated aptamer and zwitterionic peptide (EKEKEKE-PPPPC) on macroporous gold-coated GCE.The combined use of macroporous gold and anti-fouling peptides imparted higher surface area and improved anti-fouling properties to the prepared electrodes, resulting in a very low LOD of 42 fg mL −1 in buffer and recovery of 97.8%-101.2% of IgE in 5% FBS samples compared to IgE spiked in the samples. [133]Wang et al. reported an electrochemical assay for microRNA24 (miRNA24) detection in serum samples using a conductive polymer surface immobilized with an anti-fouling peptide.Cysteine-containing CTHNDRKQE zwitterionic peptide and RNA-specific thiolated DNA was covalently immobilized to the amine group of PANI electropolymerized on a GCE using succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as the conjugation agent.The developed platform outperformed commonly used anti-fouling materials such as PEG and MCH and detected miRNA24 with a LOD of 3.1 fM in buffer; the prepared biosensor also showed a <5% difference of miRNA24 concentrations detected in 5% human serum samples compared to miRNA24 concentrations measured using fluorescence quantitative PCR. [134]Similarly, Wang et al. reported the electrochemical detection of IgE in serum using IgE-specific aminated aptamer and CHHHDDD peptide, both of which were covalently immobilized sequentially with carboxyl groups present on electrodeposited PABA-coated GCE using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/ Nhydroxysuccinimide (NHS) chemistry.The prepared biosensor reported a LOD of 0.52 pg mL −1 in buffer and recovered 97.8%-100.0% of IgE in 10% FBS samples compared to IgE spiked in the samples. [135]However, it is again notable that all of the physiolog-ical fluid results reported in this context must be heavily diluted for successful analysis.
Co-immobilization of the peptide and DNA on the electrochemical sensor surface may compromise the sensitivity and anti-fouling performance of the biosensor due to competition between probe and peptides.As an alternative, a peptide-DNA conjugate (P2-DNA) was reported by Chen et al. in which a biotinylated anti-fouling peptide bearing an azide group (P2) was attached to a dibenzocyclooctyne (DBCO)-containing aptamer (DNA) to form a conjugate.This peptide-aptamer conjugate was then immobilized on PEDOT/AuNPs electrodeposited GCEs pre-functionalized with a streptavidin-linked biotinylated peptide (P1) to form a P2-DNA/streptavidin/P1/AuNPs/PEDOT/GCE biosensing platform for the detection of CA125 in undiluted serum. [136]Peptides can also be used in a click-chemistry based reaction with a biorecognition element as reported by Chen et al., in which an anti-fouling peptide functionalized with both azide and biotin groups was attached to DBCO-modified CEA specific antibody (via the azide group) and to streptavidin-coated electrodes (via the biotin group).The prepared platform was able to detect CEA using DPV with a LOD of 40 fg mL −1 in buffer and showed a 4.6% difference in the measured CEA concentration in clinical serum samples between the prepared biosensor and the standard ECL assay. [137]The functionality of both these examples in undiluted serum suggests clear benefits in integrating the anti-fouling and detection entities together into a single functional unit.
The flexibility inherent in the design of the peptides allows for the rational engineering of electrode coatings to address multiple detection challenges.For example, the negative charge  [141] Copyright 2022, Elsevier.
of DNA in aptamer-based probes can attract positively charged foulants and thus reduce the activity of an electrochemical biosensor in physiological fluids.To address this challenge, Li et al. developed an electrochemical assay based on an electroneutral peptide nucleic acid (PNA) as a DNA-mimicking biorecognition element coupled with the neutral anti-fouling peptide (CPPPPEKEKEKEK) for the detection of SARS-CoV-2 viral RNA in human saliva.No signal loss was detected in saliva compared to the signal measured in the buffer, [138] showing the general potential of this approach.Multifunctional peptides can also be designed to include amino acids that can act as dopants for coelectropolymerization with PEDOT.For example, Han et al. designed a peptide containing three separate amino acid sequences that can act as a dopant for conducting polymers during electropolymerization, facilitate anti-fouling, and promote electrode surface attachment, [139] while Wang et al. reported a similarlyinspired multifunctional peptide with anti-fouling (AEAKAEAK), linking (PPPP), doping (DSDS) and anchoring (C) sequences on PEDOT/GCE biosensing platform for the detection of miRNA-21 in serum. [140]More recently, Zhang et al. reported using multifunctional peptide-sodium alginate (SA)-graphene oxide (GO)-Pb 2+ crosslinked gel as an anti-fouling electrochemical biosensor for the detection of matrix metalloproteinase 7 (MMP-7), a biomarker for colon cancer.The multifunctional peptides on the SA-GO-Pb 2+ -modified electrodes were coupled with pyrrole (Py) doped-urease@zeolite imidazole frameworks (urease@ZIF-Py).In the absence of MMP-7, the urea added to the biosensor produces CO 2 after its decomposition by urease enzyme, leading to PbCO 3 precipitation on the surface and resulting in a decrease in current signal; in the presence of MMP-7, the recognition se-quence in the peptide-containing urease@ZIF-Py is released to suppress PbCO 3 precipitation and thus induce minimal if any change in the current signal (Figure 9A).The prepared electrode setup exhibited effective anti-fouling properties, with 89.3% of the signal retained after incubating the electrodes in 50% serum (Figure 9B); consequently, detection of as little as 24.34 fg mL −1 MMP-7 in buffer could be achieved with a linear response range from 1 pg mL −1 to 100 ng mL −1 and 99.2%-110.7%recovery of MMP-7 in clinical human serum compared to MMP-7 spiked samples (Figure 9C). [141]Finally, Song et al. prepared a DNAdifunctional peptide conjugate in which the peptide ((2-azido)-KNQEKNQEDHWRGWVA) was designed with both anti-fouling and target recognition capabilities.The anchoring DNA interacted with AuNPs, while the peptide specifically bound to the target protein, human immunoglobulin G (IgG).The resulting biosensor demonstrated improved anti-fouling properties, enabling the direct detection of IgG in real human serum with a wide linear range (0.1 ng mL −1 to 10 μg mL −1 ), a low LOD of 0.037 ng mL −1 in buffer, and low (2.3%-11.3%)differences in IgG concentrations detected in clinical human serum samples compared to readings obtained using the immunoturbidimetry method. [142]However, it should be noted that the complex assembly of many of these multi-functional peptides may limit their scalability in practical use, despite the very promising performance metrics achievable.
Combinations of peptides with other anti-fouling polymers can also offer improved anti-fouling properties by leveraging the benefits of multiple anti-fouling mechanisms.For example, Yang et al. constructed an interface combining PEG and the anti-fouling peptide CPPPPKSESKSESHLTVSPWY on a Figure 10.A) Aptamer-immobilized F50-C50 electrodes used for in vivo detection of kanamycin in rats and the signal deviation observed during measurement in comparison to PEG-coated electrodes.Reproduced (Adapted) with permission. [153]Copyright 2022, John Wiley and Sons.B) % R CT change of IgG imprinted hydrogel film (IgG-MIH) at different concentrations of IgG in presence of 10% FBS in comparison with a non-protein imprinted hydrogel film (NIH). [162]Reproduced (Adapted) with permission.Copyright 2021, Elsevier.C) Amperometric response of a thermoresponsive gel in the presence of a fixed concentration of glucose as a function of temperature within a heatable screen-printed electrode.Reproduced (Adapted) with permission. [163]opyright 2015, American Vacuum Society.
PEDOT-AuNP-modified electrode.Effective anti-fouling properties were observed in complex biofluids such as blood and serum, enabling the accurate detection of HER2 across a wide concentration range (1.0 pg mL −1 to 1.0 μg mL −1 ) with an ultralow LOD of 0.44 pg mL −1 in buffer and a 1.4%-9.1% difference in detected readings of HER2 in clinical human serum samples compared to readings obtained from ELISA.This dual anti-fouling approach represents a potentially promising strategy for developing practical sensing platforms compatible with complex biological environments. [143]he biocompatible and tunable nature of peptides makes them highly beneficial in electrochemical biosensing.However, peptides are significantly more expensive than other types of anti-fouling polymers and can undergo oxidative or protease degradation in physiological fluids, degradation that may be enhanced in the presence of electric fields used for electrochemical biosensing. [20,144]Peptidomimetic polymers may address some of these drawbacks; however, to our knowledge, none have yet been used for electrochemical biosensing. [145]

Other Polymer-Based Anti-Fouling Materials
Beyond the PEG, zwitterionic, and peptide-based coatings most typically reported, a variety of other materials has been tested for promoting anti-fouling in electrochemical biosensing.The following sections will briefly review the most promising materials in this context, with the performance of these materials in the context of anti-fouling electrochemical biosensing summarized in Table 2.

Hydrogels
Hydrogels, networks of highly water-binding polymers with a defined pore size and capacity for water retention, offer obvious benefits in the context of electrochemical biosensing given their capacity to both bind water and control access of larger molecules (like protein foulants) to the electrode surface based on the gel mesh size.Several examples of the use of hydrogels for biosensor coatings have been reported.Chan et al. screened 172 combinatorial copolymer hydrogel materials for this purpose, identifying F50-C50 (a combination of hydroxyethylacrylamide (F) and diethylacrylamide (C) prepared by using photopolymerization) as the best anti-fouling coating for enabling real-time in vivo biosensing of kanamycin in rats using SWV (Figure 10A). [153]Another acrylamide-based anti-fouling biosensor for quantum sensing on diamond surfaces was reported by Kumar et al.; however, it has not been used for electrochemical biosensing. [154]Chen et al. reported a biomimetic hydrogel based on guanosine designed to mimic the water-soluble polysaccharides that fish naturally release to create a protective mucus layer, created by the self-association of macrocyclic tetramers in the presence of alkali metal ions and stacking through hydrogen bonding.The hydrogel was immobilized on a cationic poly(dimethyl diallyl ammonium chloride) (PDDA) layer that was electrodeposited on a GCE.PANI-polythionine hydrogel [ 98] Electropolymerization Amperometry and SWV CA125 using antibody PANI, 3-aminophenylboronic (APBA) and PVA conductive supramolecular polymer hydrogel (CSPH) [ 99] Electropolymerization and cross-linking with hydroxyl groups of PVA DPV Thrombin using capture aptamer and signal aptamer 6% reduction in current signal after incubation in 100% FBS (for 30 min).Combinations of polyacrylamide hydrogels on gold wire [ 153] Photopolymerization using lithium phenyl-  DNA-Peptide/conductive polyaniline hydrogel/cellulose nanocrystals (CNCs) on GCE [156] Precursor solution containing aniline, aminobenzenboric acid, Physiological Fluid (Human serum heated, centrifuged, and diluted to 2%): Recovery of 99.5%-106% of signal compared to spiked samples PANI-HA hydrogel on GCE [157] Free radical polymerization DPV IgG using peptide 10% signal suppression when incubated with 2 mg mL −1 HSA (for 1 h).BSA/multi-walled CNTs (MWCNTs)/glutaraldehyde (GA) on carbon electrodes [ 158] Physical adsorption

CV and EIS
Cytokine IL-6 using antibody ≈7% sensitivity loss after 1 month of exposure to either 1% BSA, unprocessed human serum or plasma.

Not reported
Thermoresponsive polymer I with pH responsive redox-polymer II and enzyme on gold electrode [ 163] UV-induced polymerization Amperometry Glucose using enzyme

Not reported
Not reported

Not reported
Adv. Sensor Res.2024, 3, 2300170 The resulting sensor was used for the detection of Tau-protein in serum samples using Tau-antibodies immobilized on the hydrogel using EDC/NHS chemistry, enabling Tau-protein detection with an LOD of 1.31 pg mL −1 in buffer with effective anti-fouling properties against serum and different concentrations of BSA and 1% FBS.The prepared biosensor recovered 92.7%-106.2% of Tau-protein in 10% human serum compared to Tau-protein spiked in the sample. [155]o address the drawback of the insulating nature of the hydrogel coatings (directly parallel to the challenges with noncrosslinked polymeric coatings), conductive hydrogels have been prepared to improve charge transport and thus detection sensitivity; the crosslinked network can help entrap and/or maintain the orientation of conductive fillers to improve electrode stability.For example, He et al. reported a conductive hydrogel consisting of PANI, cellulose nanocrystals (CNCs), and polyvinyl alcohol (PVA) crosslinked with aminobenezenboric acid, leveraging boronate formation between boronic acid and the hydroxyl groups of PVA.The hydrogel showed negligible fouling when incubated with different proteins such as BSA, -lactoglobulin, lipoprotein lipase or with serum due to the combined effects of the anti-fouling peptide and the highly hydrophilic hydrogel layer.Following attachment of an anti-fouling peptide (EKEKEKEK) linked with an ATP-binding aptamer, ATP detection with a LOD of 0.025 pM in buffer and a recovery of 99.5%-106% (relative to an ATP-spiked sample) in processed (heated and centrifuged) and 50-times diluted human serum samples could be achieved. [156]Using a complementary approach, Liu et al. employed the functionality of boronic acid for PVA and PANI crosslinking to form a polyaniline-polyvinyl alcohol hydrogel (PPH) coating with high specific capacitance and stability that could detect IgG with a broad detection range (0.1 ng mL −1 to 10 μg mL −1 ) and a low LOD (0.043 ng mL −1 ). [157]SA-based 3D porous and conductive polymeric hydrogels have also been reported for electrochemical biosensing in complex samples.For example, Li et al. reported a hydrogel film formed from BSA and multi-walled CNTs (MWCNTs) crosslinked with glutaraldehyde (GA) for the detection of glycated haemoglobin A (HbA1c) in human serum samples using EIS.The prepared electrodes retained 88% of the CV signal after 1 month of exposure in human serum and detected HbA1c with a LOD of 0.4% (0.8 μg mL −1 ) in unprocessed human serum. [158]Li et al. reported GA cross-linked amyloid-like BSA coatings on PANI-modified electrodes for the detection of IgM in human serum samples.The coated electrodes showed a <5% change in DPV signals after exposure to 100% human serum and human blood for 30 min, enabling the detection of IgM with a LOD of 2.32 pg mL −1 in buffer and a similar LOD observed in 100% FBS. [159]Similarly, Sabate del Rio et al. reported a BSA-GA-gold nanowire nanocomposite hydrogel coated with IL-6 antibodies that could retain >88% of the CV signal after 1 month of exposure to unprocessed human plasma, enabling IL-6 detection with a LOD of 23 pg mL −1 in unprocessed human plasma. [160]Au and Ag nanoclusters have also been encapsulated in BSA-based hydrogel coatings for electrochemiluminescence biosensing of glutathione (GSH) in serum by Han et al., but electrochemical biosensing as not yet been reported. [161]ile hydrogels show exceptional anti-fouling properties (particularly after long-term storage in physiological fluids), the key challenge for using hydrogels as coatings for electrochemical biosensors comes in controlling the thickness of the hydrogel on the electrode surface to counteract the electrically insulative nature of hydrogels.Designing methods to create more consistent very thin hydrogel films may unlock the potential of hydrogels in the context of electrochemical biosensor coatings.

Molecularly Imprinted Polymers
Molecularly imprinted polymers (MIPs) are polymers formed by the polymerization of monomers in the presence of a target analyte to be detected; following polymerization, the target analyte is removed to form cavities complementary to the target analyte. [164]elative to other sensing platforms, MIPs are known for their high specificity and stability for recognizing specific target analytes based on their ability to bind their target not only based on physicochemical interactions but also by shape.However, the high internal surface area of MIPs coupled with the sensitivity of their target binding to the shape and accessibility of the target cavity make them particularly susceptible to biofouling. [162]s such, significant effort has been invested in designing MIPs with anti-fouling properties that are suitable for electrochemical biosensing.For example, Hu et al. reported the electrochemical detection of the mycotoxin zearalenone (ZEA) by electropolymerizing methylene blue and an anti-fouling 1-aminoethly-3methylimidazolium nitrate ([APMIm]NO 3 ) ionic liquid in the presence of ZEA on the biosensor surface using CV, with the ZEA later removed using a methanol-acetic acid mixture.The prepared MIP-based electrochemical biosensor enabled the detection of ZEA with an LOD of 12.7 nM in buffer and recovered 90%-112% of ZEA in human serum samples compared to spiked ZEA in serum. [165]Similarly, Turco et al. electropolymerized an anti-fouling polydopamine MIP on a gold surface using CV in the presence of sulfamethoxazole as a target analyte; following removal of the sulfamethoxazole template using 5% acetic acid, the resulting biosensor showed no significant fouling when incubated in milk samples and sulfamethoxazole detection was enabled with an LOD of 800 nM in buffer and recoveries of 99±8% and 103±6% in two sulfomethoxazole-spiked and processed (precipitated, filtered and diluted) semi-skimmed milk samples. [164]As such, incorporating anti-fouling approaches can significantly improve the consistency of MIP-based electrochemical biosensor readings in real physiological fluids and foods.
Anti-fouling protein-imprinted hydrogels prepared through similar molecular imprinting technology have also been successfully applied for the electrochemical biosensing of proteins.For example, Cui et al. reported a protein-imprinted hydrogel prepared by room-temperature free radical polymerization of acrylamide, N-isopropylacrylamide (NIPAM), the crosslinker N,Nmethylenebis(acrylamide), IgG as the template protein, ammonium persulfate (APS) as the initiator, and tetramethylethylenediamine (TEMED) as the promoter.The presence of the temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) facilitates easy removal of IgG protein at higher temperature (37°C) due to deswelling of the PNIPAM chains and thus steric exclusion of the template from the MIP; IgG protein binding affinity can be recovered when the temperature is again lowered temperature (20 °C).The resulting biosensor showed only a 5.2% increase in R CT after incubation with 25% FBS for 24 h, enabling the detection of IgG with a LOD of 0.03 ng mL −1 in both buffer and 10% FBS (Figure 10B). [162]The same group recently reported dual-responsive and anti-fouling protein-imprinted polymers for the electrochemical detection of CD44 biomarker in serum samples using an alginate gel-based MIP and hyaluronic acid-coated screen printed electrodes (SPEs).High specificity toward the CD44 target (LOD of 1.41 × 10 −5 ng mL −1 for the MIP-based biosensor and 1.51 × 10 −6 ng mL −1 for the HA-based biosensor in buffer) but negligible interference from non-specific adsorbed proteins tested at a concentration 100 times higher than that of CD44 were observed. [166]A similar report of using an anti-fouling molecularly imprinted alginate gel for the electrochemical detection of HSA in 20% FBS showed analogous useful anti-fouling properties (≈5% change in R CT after incubation in 20% FBS for 30 days) and detection capacity (LOD of 0.03 × 10 −6 ng mL −1 in a similar LOD in 20% FBS). [167]However, it is noteworthy that none of these examples reported effective sensing in undiluted serum.
Nanocomposite MIP design strategies have also been explored to enhance the conductivity of the MIP to promote effective charge transfer and thus enhanced sensitivity in complex biofluids.Ghaani et al. reported a molecularly imprinted polymercoated and multi-walled carbon nanotubes-modified glassy carbon electrode (MIP/MWCNTs/GCE) for the detection of 4,4′methylene diphenyl diamine (MDA) in which the MWCNT coating was designed to enhance conductivity and thus sensor sensitivity.Following the electrodeposition of a chitosan nanoparticle film onto the MWCNTs/GCE surface in the presence of MDA as a template using cyclic voltammetry and subsequent removal of the MDA template, an MDA detection limit of 15 nM could be achieved with a linear concentration response over the range of 0.5-100 μM.The proposed sensor was effectively employed for the determination of leached MDA concentrations in packaging bags filled with food simulant solution followed autoclaving, achieving satisfactory recoveries ranging from 94.1% to 106.8%. [168]Cui et al. used dopamine self-polymerization to create an MIP consisting of conductive graphdiyne, anti-fouling PEG, and coating-enabling polydopamine in the presence of Creactive protein (CRP), followed by removal of CRP using acetone.The prepared MIP showed high-antifouling (≈5% change in R CT upon incubation in 10% FBS for 30 min) and detected CRP with a LOD of 0.41 × 10 −5 ng mL −1 in both buffer and 10% FBS. [169]iorecognition probes can also be added into MIP-based coatings, working together with the shape-specificity of the MIPs to further improve biosensor specificity.However, care must be taken that the addition of such probes does not significantly alter the size, shape, or interfacial chemistry of the MIP binding cavity.For example, Jolly et al. reported an aptamerfunctionalized molecularly imprinted polymer based on polydopamine for the EIS-based electrochemical detection of PSA.The prepared biosensor was three-fold more sensitive than normal aptasensors and showed the least resistance change to incu-bation against human serum albumin, enabling PSA detection with a LOD of 1 pg mL −1 in buffer, however, PSA detection in a physiological fluid was not reported. [170]

Stimuli-Responsive Polymers
Polymers responsive to physical stimuli such as temperature, pH, etc., have been prepared and applied for electrochemical biosensing, taking advantage of reversible phase transitions to alter the interfacial hydrophobicity, thickness, and/or probe steric availability to suppress fouling while maximizing binding efficiency.For example, Bunsow et al. reported a thermoresponsive hydrogel layer formed by copolymerizing thermoresponsive PNIPAM and anti-fouling OEGMA via electrochemically-induced free radical polymerization for the amperometric detection of glucose using glucose oxidase enzyme trapped within the polymeric hydrogel layer.The prepared hydrogel-coated electrodes showed high resistance to HSA adsorption for 60 min while enabling hydrogel composition and temperature-specific tuning of the sensing current due to changes in the mesh size of the hydrogel (and thus the accessibility of glucose to entrapped glucose oxidase) as a function of hydrogel swelling/deswelling (Figure 10C). [171]imilarly, electrochemical co-deposition of the thermoresponsive polymer poly(-ethoxytriethylenglycol methacrylateco-3-(N,N-dimethyl-N-2-methacryloyloxyethyl ammonio) propanesulfonate-co--butoxydiethylenglycol methacrylateco-2-(4-benzoyl-phenoxy)ethyl methacrylate)) together with the pH-responsive redox polymer (poly(glycidyl methacrylate-coallyl methacrylate-co-poly(ethylene glycol) methacrylate-co-butyl acrylate-co-2-(dimethylamino)ethyl methacrylate)-[Os(bpy) 2 (4-(((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)methyl)-N,Ndimethylpicolinamide)] 2+ ), pyrroloquinoline quinone-soluble glucose dehydrogenase (GDH) enzyme, and 2,2′-(ethylenedioxy)bis-(S-acetylethanthiol) cross-linker on a heatable gold screen printed electrode was used to create an anti-fouling gel for the amperometric detection of glucose.Above the phase transition temperature, the gel collapsed to both protect the enzyme from degradation over storage and slow glucose diffusion toward the enzyme active site, resulting in an off sensing state; upon cooling, the gel re-swells to promote glucose diffusion into the coating and activate anti-fouling properties. [163]espite the interesting physical properties of stimuliresponsive polymer-coated sensors, it remains to be seen whether such coatings can practically improve biosensor performance in real applications.Upper critical solution temperature coatings in which the gel is collapsed upon cold storage (to protect an enzyme) but swells upon exposure to physiological fluids (to facilitate diffusional probe access while activating hydration-based anti-fouling) may offer more specific practical benefits for sensing compared to lower critical solution temperature temperatures such as PNIPAM, although such benefits have yet to be demonstrated.
Enzyme triggering has also been explored.Meißler et al. reported an enzyme-triggered peptide-PEG-based anti-fouling layer on a titanium surface, which was tested for anti-fouling using QCM-D. [172]However, this reported coating was not used for electrochemical biosensing, nor is the potential utility of enzyme triggering in electrochemical biosensing applications particularly clear. [173]

Continuing and Emerging Challenges in the Design of Anti-Fouling Polymer/Peptide-modified Electrochemical Biosensors
Despite the multiple advances made in improving the antifouling and thus sensing performance of electrochemical biosensors, there remains a need to develop versatile electrode coating materials that can be 1) integrated into sensor more easily, 2) improve the detection of specific targets, 3) better resist fouling, particularly with high surface area electrodes, 4) enhance signal magnitude to enable lower LODs in physiological fluids, and 5) remain more stable over time, both in the context of storage as well as active use.Below we summarize current research to address key outstanding challenges in the design of anti-fouling electrochemical biosensors (e.g., stability, conductivity, and hydration) in addition to emerging challenges such as the promotion of self-healing and achieving flexibility.

Stability
The redox environment to which electrochemical biosensor coatings are exposed creates challenges in stabilizing many coatings over repeated or long-term operation times.PEG-based materials are particularly prone to oxidative damage, [178] the branched POEGMA materials appear to at least partially address the challenge.Alternately, blends of PEG with other polymers have been explored.For example, blending chitosan with PEG has been reported to enhance PEG stability by leveraging chitosan's improved stability and film-forming ability.Mahmoud et al. designed a glutathione (GSH) sensor coated with chitosan, PEG, porous magnesium metasilicate (PMMS) (a Cu(II) adsorbent), and silver nanoparticles (AgNPs, for enhanced conductivity).Utilizing Cu(II) electrocatalysis from Cu(II)-PMMS, the sensor detected GSH without added Cu(II) with a dynamic range of 0.1 to 125 μM and a low detection limit (0.03 μM) in both dietary supplements and human plasma serum samples. [146]an et al. a similar additive stabilization approach to develop a photoelectrochemical (PEC) immunosensor using a polyacrylic acid (PAA)/PEG anti-fouling coating on a ZnO/CdSe semiconductor composite to detect neuron-specific enolase (NSE) with high sensitivity and precision, resulting in a wide linear range (0.10 pg mL −1 to 100 ng mL −1 ) and a low detection limit (34 fg mL −1 ). [179]onventional peptides can also exhibit varying degrees of susceptibility to degradation depending on their specific sequence, structure, and environmental conditions, resulting in often poor stability and degradation in complex biological samples (particularly in the redox environment of electrochemical biosensing). [148]Multiple approaches have been investigated to improve anti-fouling peptide stability in this context.Yang et al. designed peptides incorporating -aminoisobutyric acid (Aib), an artificial amino acid that demonstrated resistance to enzymatic degradation in human serum.The designed Aib-peptide also contained a cysteine residue at one end to enable anchoring of the peptide on the PEDOT/AuNP substrate and another anti-fouling sequence at the other end that can bind to IgG.The introduction of Aib residues provided resistance to proteolytic degradation in complex biofluids, enabling enhanced sensitivity for electrochemical sensing in human sera. [148]Zhao et al. took a complementary approach to stabilize zwitterionic anti-fouling peptide domains based on glutamic (E) and lysine (K) residues against enzymatic degradation by replacing the vulnerable -K with the more stable synthetic -K.This engineered /-peptide exhibited significantly improved stability relative to conventional peptides composed solely of -amino acids while enabling IgG sensing with a wide linear detection range from 100 pg mL −1 to 10 μg mL −1 and a low detection limit of 33.7 pg mL −1 (S/N = 3). [149]Han et al. took an alternate approach by designing a cyclic peptide (CEKEKEKEK) that demonstrated similar anti-fouling benefits to the linear peptides NH 2 -CEKEKEKEK-COOH and NH 2 -CPPPPEKEKEKEK-COOH but with significantly enhanced enzymatic resistance.This peptide was employed to detect the receptor binding domain (RBD) of SARS-CoV-2 antigen in human biofluids over concentrations ranging from 1.0 pg mL −1 to 100.0 ng mL −1 with an impressive limit of detection at just 0.45 pg mL −1 , a result that outperformed ELISA kits particularly in high-concentration human blood (Figure 11A). [150]Hu et al. similarly manipulated peptide morphology by fabricating a biomimetic tree-like multifunctional anti-fouling peptide (TMAP), with the multivalent interaction of the peptide showing enhanced PD-L1 (programmed death ligand) exosome binding, effective anti-fouling properties, and improved stability.Coordination of Zr 4+ to the exosome membrane facilitates exosome decoration with silver nanoclusters (AgNCs); upon exosome binding to the TMAP binding sequence, the Zr 4+ and thus the AgNCs are released to facilitate low detection limits of 296 PD-L1 exosome mL −1 in serum while supporting a wide dynamic range (78 to 7.8 × 10 7 particles mL −1 ) appropriate for clinical diagnostic use (Figure 11B). [151]Similarly, Xu et al. integrated an engineered branched peptide (EBP) featuring an inverted Y-shaped structure with anti-fouling branches, providing high resistance against biofouling and improved degradation resistance compared to linear peptides and thus enabling the detection of the cardiac troponin I (cTnI) biomarker in human serum. [152]hile zwitterionic materials exhibit generally higher chemical stability compared to PEG and peptides, their anti-polyelectrolyte properties and varying interactions with salt can pose significant solution stabilization challenges that can either induce deswelling of the coating (reducing water binding and thus anti-fouling properties) or desorption of the coating from the biosensor surface.Co-polymerization with anchoring monomers offers one strategy to improve anchoring of zwitterionic coatings on an electrode surface.For example, Yang et al. electropolymerized polyaniline on a GCE (PANI/GCE) followed by performing a hydrophobic interaction-based copolymerization (PSN) of zwitterionic sulfobetaine methacrylate (SBMA) with NIPAM above the PNIPAM) phase transition temperature, resulting in the creation of an anti-fouling biosensor coated with a stable self-assembled monolayer.The biosensor was capable of directly detecting CA125 in undiluted serum over a concentration range of 0.01-1000 U mL −1 with a detection limit of 2.7 mU mL −1 while also showing sustained 15-day anti-fouling in buffer (Figure 11C). [147]However, methods to improve the chemical stability of zwitterionic anti-fouling coatings are also under Figure 11.A) Anti-fouling cyclic peptide on gold nanoparticle (AuNP) electrodeposited PEDOT/glassy carbon electrodes (GCEs) exhibit exceptional antifouling properties, enabling sensitive and selective electrochemical detection of COVID spike glycoprotein (receptor binding domain, RBD) using the ACE2 receptor in blood with ELISA-level accuracy.Reproduced (Adapted) under the terms of the CC BY-NC 3.0 DEED license. [150]Copyright 2023, Royal Society of Chemistry.B) A biomimetic tree-like branched multifunctional anti-fouling peptide-based electrochemical sensor achieved highly selective and sensitive detection of programmed death ligand (PD-L1) exosomes in serum, utilizing zirconium assisted (Zr 4+ ) silver nanocluster (Ag-NC-COOH) release from exosomes.Reproduced (Adapted) with permission. [151]Copyright 2023, Elsevier.C) Hydrophobic interaction-based copolymerization of zwitterionic sulfobetaine methacrylate (SBMA) and N-isopropylacrylamide (NIPAM) on a polyaniline (PANI)-modified glassy carbon electrode (PANI/GCE) to create a stable, anti-fouling aptasensor with excellent conductivity that allows for the direct detection of cancer antigen 125 (CA125) in undiluted serum.Reproduced (Adapted) with permission. [147]Copyright 2023, Elsevier.D) An electrochemical biosensor based on a zwitterionic peptide hydrogel with a cysteine terminal for facile antibody immobilization on gold surfaces and modified PEDOT electrodes that demonstrates effective anti-fouling properties and prostate-specific antigen (PSA) detection.Reproduced (Adapted) with permission. [175]Copyright 2023, Elsevier.(Created with BioRender.com).
development to further improve the robustness of zwitterionic coatings in long-term sensing applications.For example, Xu et al. combined a conjugated polythiophene backbone, a multifunctional zwitterionic side chain, and a mesogenic unit to create a liquid crystalline mesophase-based coating that showed significantly enhanced stability while maintaining (or even improving) anti-biofouling properties and coating conductivity. [180]

Conductivity
The low conductivity of inherently insulating polymer coatings remains a significant bottleneck to achieving high signal:noise ratios in electrochemical biosensors, limiting signal transduction and leading to reduced sensitivity.Many examples of combining anti-fouling polymers or peptides with conductive nanoparticles (most typically metal nanoparticles or conjugated carbon-based nanoparticles) have already been discussed in this review to address this problem, [122,141,143] although emerging approaches tend to select nanoparticle fillers with multiple beneficial properties beyond conductivity.For example, Zhou et al. developed a nanocomposite coating offering not only enhanced conductivity but also anti-corrosion and anti-bacterial properties by modifying an acrylic resin (PAZ/N-PMI) with zinc ions and a structural functional monomer (N-phenyl maleimide) and incorporating a conductive nanofiller (F-MWCNTs-OH@SiO 2 ).The resulting composite coating exhibited >99.98% effectiveness in killing both Staphylococcus aureus and E. coli in addition to improved corrosion resistance. [181]ngineering of the physical coating properties can also promote improved inherent conductivity even without conductive fillers.For example, Kim et al. designed ferrocene-based amphiphilic PEG methacrylate conjugated layer-by-layer coatings with precise control over layer thickness and effective anti-biofouling properties that demonstrated improved electrical signals and thus enhanced sensitivity relative to other PEG-based coatings. [182]Alternately, Poisson et al. designed cationic bottlebrush brush polymers via a two-step process involving surfaceinitiated ring-opening metathesis polymerization (SI-ROMP) followed by surface-initiated activators regenerated by electron transfer atom transfer radical polymerization (SI-ARGET ATRP), allowing for independent control of the degree of polymerization of the backbone and the side chains of the bottlebrush structure.The resulting optimized bottlebrush polymers exhibited a coating with a 70% increased thickness, but an improved ionic conductivity compared to conventional linear polymer brushes, a result attributed to the higher density of the bottlebrush polymers relative to linear polymers. [183]

Hydration
Maintaining as dense as possible a hydration layer has been linked to effective anti-fouling properties. [184]As such, any strategy that can enhance surface hydration in principle should also improve anti-fouling.In this context, zwitterionic peptide hydrogels are attracting increasing interest given that they can combine the high water hydration capacity of zwitterions with the tunable chain densities (controlled by the crosslink density) of a hydrogel.Combining this approach with conductive nanoparticles has been demonstrated as a promising strategy for promoting effective electrochemical biosensing.For example, Liu et al. developed a zwitterionic peptide hydrogel based on the sequence FFCCEKEKEK, which features a fluorene methoxycarbonyl group modified N-terminal, that could maintain a dense hydration layer via ionic solvation and can self-assemble on conductive AuNPs via Au-S bonds using the cysteine residues in the peptide.The resulting hydrogel enhances the electroactive area and facilitates electron transfer while promoting effective anti-fouling, enabling dopamine (DA) detection with a LOD of 0.12 nM, a broad linear range from 0.2 to 1900 nM, and high selectivity. [174]Using a similar strategy, Wang et al. developed an electrochemical biosensor for detecting PSA in human serum using a zwitterionic peptide hydrogel based on the sequence CFE-FKFC on PEDOT-modified electrodes, again using cysteine-gold interactions; the resulting biosensor demonstrated effective antifouling properties, a linear response range of 0.1 to 100 ng mL −1 and a low LOD of 5.6 pg mL −1 (Figure 11D). [175]Developing methods to fabricate thinner and more conductive zwitterionic hydrogels without compromising their anti-fouling properties would be anticipated to even further improve performance.

Self-Healing
Mechanical or chemical damage to sensors with anti-fouling coatings can negate the benefit of the anti-fouling coating while also creating redox gradients across the electrode that may lead to enhanced electrode instability.In this context, the development of anti-fouling coatings that can also self-heal is likely to lead to biosensors with enhanced performance lifetimes in complex fluids.Self-healing polymer coatings can autonomously repair minor physical damage that may occur during sensor operation, promoting sustained sensor performance over an extended operational lifespan that ultimately reduces the need for frequent recalibration or sensor replacement as well as the high environmental footprint of disposable sensors. [185]ost self-healing examples to-date with specific anti-fouling properties involve hydrogels.Qiao et al. developed an antibiofouling polypeptide complex hydrogel (AuNPs/MoS 2 /Pep hydrogel) with self-healing properties and suitable electrochemical performance for the continuous monitoring of uric acid and ascorbic acid levels in sweat.The polypeptide sequence was engineered to contain both a domain rich in hydrophilic groups (facilitating anti-fouling) as well as naphthalene groups that can participate in - stacking interactions with the MoS 2 filler (enabling self-healing) (Figure 12A). [176]nherent self-healing properties can also be introduced by exploiting crack-activated hydrogen bonding, as demonstrated by the polyvinyl alcohol/sulfuric acid (PVA/H 2 SO 4 ) hydrogel electrolyte reported Ma et al. that could self-heal based on dynamic hydrogen bonding between PVA chains.This hydrogel electrolyte was used to assemble a multifunctional flexible supercapacitor with high energy density and low interfacial contact resistance, although the anti-fouling properties were not explicitly tested. [186]Alternately, Badawi et al. reported a self-healing hydrogel electrolyte composed of sodium alginate and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) that not only exhibited rapid self-healing capabilities but also a high specific capacitance ideal for rapid and efficient charge transfer while enhancing the sensor's longevity and reliability. [187]However, given that self-healing requires the presence of highly labile bonds that also tend to reduce the mechanics of the ultimate coating, significant research opportunities remain in this area to balance the speed of, and conditions amenable to, self-healing without compromising the conductivity, hydration degree, or environmental stability of the coating.
Other self-healing polymers have also been incorporated into electrochemical biosensing platforms that, while not explicitly anti-fouling, offer effective signal detection and thus inspiration for self-healing anti-fouling coating design.For example, Wang et al. used polyurethane (PU) and 2-formylbenzeneboronic acid (2-FPBA) coupled with MXene and graphene oxide (GO) to create a self-healing elastomeric substrate that exhibits selfhealing at room temperature via dual dynamic imine-borate ester covalent crosslinks and photothermal conductivity.The constructed microfluidic electrochemical biosensing platform enabled the detection of exosomes indicative of colorectal cancer over a wide detection range (50-10 5 particles μL −1 ) with a low LOD (42 particles μL −1 ), making it a disposable, reusable, costeffective, and rapid analysis platform. [188]Son et al. created a selfhealable and conductive ink by incorporating graphene into the self-healing polymer poly(1,4-cyclohexanedimethanol succinateco-citrate), with the printed serpentine-structured electrode exhibiting good conductivity while spontaneously healing during 10 cut-heal cycles with a healing time of just 12 s at ambient conditions while retaining a sensitivity of 99% for detecting Na + in sweat in real time. [189]The incorporation of antifouling hydrogels within these of similar self-healing coatings thus offers potential, particularly in designing wearable and implantable sensors with enhanced performance.A) A self-healing anti-biofouling complex hydrogel composed of gold nanoparticle (AuNPs), molybdenum disulfide (MoS 2 ) and polypeptide integrated into an electrochemical sensor for continuous monitoring of uric acid and ascorbic acid levels in sweat.Reproduced (Adapted) with permission. [176]Copyright 2023, American Chemical Society.B) A non-invasive electrochemical wearable sweat sensor patch based on an iontophoretic hydrogel system consisting of tannic acid (for adhesion), silver (Ag), carbon nanotube (CNTs), and polyaniline (TA-Ag-CNT-PANI) capable of detecting tyrosine from sweat analysis by catalytic oxidation.Reproduced (Adapted) with permission. [177]Copyright 2023, Elsevier.C) A self-healing, stretchable anti-fouling cellulose-based conductive hydrogel composed of cross-linked polyaniline (PANI), 2,2,6,6-tetramethylpiperidine-1-oxylradical (TEMPO)oxidized CNFs (TOCNF), and PVA/borax (PVAB) for real-time electrochemical sweat analysis.Reproduced (Adapted) with permission. [190]Copyright 2022, John Wiley and Sons.(Created with BioRender.com).

Flexibility
Flexible and wearable sensors are gaining significant attention for their ability to continuously monitor biomarkers, with antifouling polymers being critical to improving accuracy of such sensors.For example, Xu et al. introduced a noninvasive wearable sweat sensing patch consisting of a nanocomposite hydrogel comprised of a tannic acid (TA) (for adhesion), silver (Ag), carbon nanotubes (CNTs), and polyaniline (TA-Ag-CNT-PANI).The hydrogel not only detected pH and tyrosine (Tyr) levels in sweat simultaneously but also used pH values to enhance the accuracy of Tyr detection by compensating for pH-related variations in Tyr responses.The inclusion of tannic acid chelated-Ag nanoparticles (TA-Ag-NPs) and carbon nanotubes (CNTs) not only enhanced the hydrogel's flexibility and conductivity (enabling accurate monitoring of Tyr levels across various sweat composi-tions) but also provided antibacterial properties.(Figure 12B). [177]s another example, Qin et al. constructed a flexible sensor by polymerizing polyaniline (PANI) in situ with oxidized cellulose nanofibrills and cross-linking with polyvinyl alcohol/borax.In addition to exhibiting fast self-healing properties (95% recovery in just 10 s, attributed to the highly reversible PVA/borax interactions), this sensor exhibits high stretchability (1530%) and conductivity (0.6 S m −1 ) to enable accurate measurement of Na + , K + , and Ca 2+ in sweat with high sensitivities (0.039, 0.082, and 0.069 mmol −1 , respectively) in real time (Figure 12C). [190]However, maintaining high hydration (as essential for promoting effective anti-fouling based on key anti-fouling mechanisms) in wearable flexible materials can be challenging given that the majority of such sensors are exposed to air.While strategies such as the incorporation of humectants (e.g., glycerol), [191,192] hydrating ions (e.g., lithium), [193,194] or co-solvents [195] have shown promise for reducing the rate of water evaporation, additional research is required to better suppress dehydration without compromising long-term sensor performance.Expanding the scope of sensing targets beyond the more small molecule/ion targets most typically reported also represents a research challenge that could expand the functionality of flexible electrochemical biosensors into different real-time sensing applications.
It should be emphasized that the fouling challenges and corresponding surface coating solutions described herein are also applicable to solving challenges in optical biosensing applications, in which interaction with foulants present in complex media can also lead to low signal-to-noise ratios (SNR) or high background signals.While the coating options available for optical sensors are often broader given that electrical/ion conductivity is not typically required (unlike with electrochemical biosensors), surface plasmon resonance (SPR)-based sensors typically use gold chips or gold-coated substrates such that thiolated anti-fouling polymers similar to those used for electrochemical biosensing can be directly applied for easy attachment to the biosensing surface.Moreover, anti-fouling polymers with the ability to attach higher densities of biorecognition elements can similarly help to increase the detection sensitivity and achieve better stability in optical biosensing applications. [196]

Conclusion
The development and integration of polymer or peptide-based anti-fouling materials and strategies has significantly advanced the practical use of electrochemical biosensors.Polymer/peptidebased anti-fouling coatings have not only enhanced the accuracy and longevity of electrochemical biosensors but have also expanded their capabilities to enable monitoring of more diverse biomarkers in a variety of complex biological environments.The successful deployment of electrochemical biosensors for rapid point-of-care diagnosis at doctor's offices or in low-resource environments, or in emerging wearable biosensor applications that can facilitate continuous monitoring of various biological signals, depends on further improving the conductivity, selectivity, and stability of current anti-fouling coatings without compromising the core performance of the biosensor.Emerging anti-fouling materials or polymers from other fields such as tissue engineering, microfluidics, organ implantation, marine anti-fouling, etc. can be adapted moving forward in electrochemical sensing applications to achieve better stability, higher sensitivity, lower needs for sample processing, higher SNRs, and lower background signals.In combination, new strategies to improve the surface adhesion of coatings to diverse substrates, reduce the coating thickness without compromising anti-fouling properties, create selfhealing coatings, and designing flexible/wearable sensors that do not compromise the essential hydration of most anti-fouling biosensors are expected to further expand the applications of such sensors in various health monitoring and environmental analysis applications.In this context, further optimization of coating deposition methods is essential to enhance the performance and longevity of diverse materials and sensing systems while improving the sustainability of both sensor manufacturing and sensor materials, particularly important to minimize waste and environmental footprint if the rapidly advancing performance and reduced cost of electrochemical biosensors results in them being more widely used in point-of-care or wearable applications worldwide.

Figure 1 .
Figure 1.Schematic summarizing the major anti-fouling mechanisms, the resulting classes of anti-fouling polymers/peptides that have been developed, and the techniques used to deposit anti-fouling polymers or peptides on electrode surfaces (Created with BioRender.com).

Figure 3 .
Figure3.A) UV-photopolymerized CNTs/acrylamide copolymer hydrogel anti-fouling film supporting a sandwich immunoassay to detect C-reactive protein electrochemically.B) Electroactive surface area (EASA) of uncoated electrodes and polymer-coated electrodes prepared with increasing concentrations of CNTs as a function of fouling time in BSA.Reproduced (Adapted) with permission.[81]Copyright 2022, American Chemical Society.

Figure 6 .
Figure6.A) PEG-coated PPy on glassy carbon electrodes (GCEs) immobilized with capture DNA (C1) as an anti-fouling electrochemical biosensor for detecting target miRNA (T2).B) Anti-fouling performance of the electrodes before and after incubation in different dilutions of human serum.C) DPV current response obtained with different concentrations of miRNA.Reproduced (Adapted) with permission.[120]Copyright 2019, Elsevier.

Figure 7 .
Figure 7. A) Anti-fouling DNAzyme-immobilized microgel magnetic beads (mMB) used for the electrochemical detection of E. coli using capture DNAcoated nanostructured gold electrodes.B) Current density obtained from mMB and commercial magnetic beads (cMB) in the presence of 10 5 CFU mL −1 E. coli in buffer and human clinical urine samples.C,D) Current density of redox DNA released from mMBs as a function of different E. coli concentrations in C) buffer and D) human clinical urine samples.Reproduced (Adapted) with permission.[123]Copyright 2022, American Chemical Society.

Figure 9 .
Figure 9. A) Mechanism behind the electrochemical detection of MMP-7 using multifunctional peptides.B) SWV current response of the prepared electrodes after incubation in different percentages of human serum.C) SWV current response as a function of different MMP-7 concentrations.Reproduced (Adapted) with permission.[141]Copyright 2022, Elsevier.

2 as catalyst 4 .
1% signal suppression in hydrogel with D-peptide after incubation in 100% sweat (for 30 min).16% and 44% signal suppression in hydrogel with D-peptide and L-peptide after incubation in undiluted sweat, respectively (for 280 h).0.02 μM to 0.11 mM in sweat (for uric acid) and 2 μM to 1.11 mM (for ascorbic acid) Physiological Fluid (artificial sweat): 0.006 μM for uric acid and 0.67 μM for ascorbic acid (continuous measurements); 4.8% deviations in uric acid readings compared with ECL in artificial sweat.

Table 1 .
Summary of electrochemical biosensors prepared using PEG-based polymers, zwitterionic polymers, and peptides.

Table 2 .
Summary of electrochemical biosensors employing polymers other than PEG, zwitterionic and peptides.