Molecular Imprinting on Nanozymes for Sensing Applications

biosensors

Review Molecular Imprinting on Nanozymes for Sensing Applications

Ana R. Cardoso 1,2,3,4,†, Manuela F. Frasco 1,2,3,†, Verónica Serrano 1,2,3,†, Elvira Fortunato 4 and Maria Goreti Ferreira Sales 1,2,3,*

1 BioMark@UC, Faculty of Sciences and Technology, University of Coimbra, 3030-790 Coimbra, Portugal; [email protected] (A.R.C.); [email protected] (M.F.F.); [email protected] (V.S.) 2 BioMark@ISEP, School of Engineering, Polytechnic Institute of Porto, 4249-015 Porto, Portugal 3 CEB, Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal 4 i3N/CENIMAT, Department of Materials Science, Faculty of Sciences and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, 2829-516 Caparica, Portugal; [email protected] * Correspondence: [email protected] or [email protected] † These authors contributed equally to the work.

Abstract: As part of the biomimetic enzyme field, nanomaterial-based artificial enzymes, or nanozymes, have been recognized as highly stable and low-cost alternatives to their natural counterparts. The discovery of enzyme-like activities in nanomaterials triggered a broad range of designs with various composition, size, and shape. An overview of the properties of nanozymes is given, including some examples of enzyme mimics for multiple biosensing approaches. The limitations of nanozymes regarding lack of selectivity and low catalytic efficiency may be surpassed by their easy surface modification, and it is possible to tune specific properties. From this perspective, molecularly imprinted polymers have been successfully combined with nanozymes as biomimetic receptors conferring selectivity and improving catalytic performance. Compelling works on constructing imprinted polymer layers on nanozymes to achieve enhanced catalytic efficiency and selective recognition, requisites for broad implementation in biosensing devices, are reviewed. Multimodal biomimetic enzyme-like biosensing platforms can offer additional advantages concerning responsiveness to different microenvironments and external stimuli. Ultimately, progress in biomimetic imprinted nanozymes may open new horizons Citation: Cardoso, A.R.; Frasco, M.F.; in a wide range of biosensing applications. Serrano, V.; Fortunato, E.; Sales, M.G.F. Molecular Imprinting on Keywords: molecular imprinting technology; nanozymes; enzyme-like activity; biosensing; biomimetics Nanozymes for Sensing Applications. Biosensors 2021, 11, 152. https:// doi.org/10.3390/bios11050152 1. Introduction Received: 8 April 2021 Enzymes are unique natural catalysts with outstanding efﬁciency and substrate speci- Accepted: 7 May 2021 Published: 13 May 2021 ﬁcity. Their use in bioanalytical methods, e.g., glucose oxidase (GOx) for glucose detection and horseradish peroxidase (HRP) for enzyme-linked immunosorbent assay (ELISA), is ex-

Publisher’s Note: MDPI stays neutral tensive, as it has always attracted research due to their tremendous potential [1]. However, with regard to jurisdictional claims in natural enzymes have limiting features linked to easy denaturation, and limited tempera- published maps and institutional affil- ture and pH ranges for optimal activity, hampering many of the foreseen applications [1,2]. iations. The disadvantages related to poor stability and reusability, along with high costs for preparation and purification, have led to efforts in designing synthetic mimics [3]. The research in this field spreads from semisynthetic approaches (e.g., genetic modification of natural enzymes) to artificial systems (e.g., cyclodextrins, metal complexes, porphyrins, dendrimers, polymers) [2,4]. These biomimetic materials are characterized by unique features and have Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. several advantages compared with natural enzymes. The artificial enzymes are low-cost, This article is an open access article have easy mass production, high stability (especially at high temperature), and long-term distributed under the terms and storage feasibility [4]. The progress in the field of nanomaterial-based artificial enzymes conditions of the Creative Commons or nanozymes has been fast since the first discovery of the unexpected peroxidase-like Attribution (CC BY) license (https:// activity of ferromagnetic nanoparticles [5]. Thus, nanozymes are considered alternatives creativecommons.org/licenses/by/ to natural enzymes and the prospective biomedical applications are vast, ranging from 4.0/). disease diagnostic and imaging to therapeutics [6,7]. Considering biosensing devices and

Biosensors 2021, 11, 152. https://doi.org/10.3390/bios11050152 https://www.mdpi.com/journal/biosensors Biosensors 2021, 11, x FOR PEER REVIEW 2 of 19

nanozymes are considered alternatives to natural enzymes and the prospective biomedical applications are vast, ranging from disease diagnostic and imaging to therapeutics [6,7]. Considering biosensing devices and the current technological advances, synergistic effects are expected to achieve ultrasensitive methods, such as colorimetric, fluorometric, Biosensors 2021, 11, 152 chemiluminescent, surface-enhanced Raman scattering, and electrochemical 2[7,8], of 19 with special emphasis on the electrochemical-based devices, which offer great advantages in terms of portability and feasibility of point-of-care use. Despite these enthusiastic perspectives,the currentpoor substrate technological selectivity, advances, low synergistic efficiency effects and are limited expected catalytic to achieve types ultrasensitive are challenges to methods,be tackled. such In asa colorimetric,biomimetic fluorometric,convergence, chemiluminescent, molecularly imprinted surface-enhanced polymers Raman (MIPs) are scattering, and electrochemical [7,8], with special emphasis on the electrochemical-based synthetic highly selective receptors, which have been integrated with nanozymes to im- devices, which offer great advantages in terms of portability and feasibility of point-of-care proveuse. the Despite desired these features, enthusiastic with perspectives, special outcom poor substratees for sensor selectivity, design. low The efficiency current and review startslimited by providing catalytic types an overview are challenges on the to composition, be tackled. In aenzyme-like biomimetic convergence,activities for molec- signal production,ularly imprintedand ability polymers to tune th (MIPs)e properties are synthetic of nanozymes. highly selective It is followed receptors, by which addressing have the basesbeen of integrated MIP technology, with nanozymes fabrication to improve methods, the and desired function features, as synthetic with special catalysts. outcomes Finally, emphasisfor sensor is given design. to The the currentmost recent review progress starts by on providing creating an MIPs overview on nanozymes on the composi- along with detectiontion, enzyme-like systems, activitiesand the forfuture signal perspectives production, andof this ability exciting to tune field the properties of research of are summednanozymes. up to It conclude. is followed by addressing the bases of MIP technology, fabrication methods, and function as synthetic catalysts. Finally, emphasis is given to the most recent progress on creating MIPs on nanozymes along with detection systems, and the future perspectives 2. Design of nanozymes of this exciting field of research are summed up to conclude. As natural enzymes are efficient biocatalysts, there has always been great interest in mimicking2. Design the of nanozymesproven high substrate specificity and superior catalytic activities. Moreo- ver, theAs development natural enzymes of synthetic are efficient approaches biocatalysts, could there overcome has always the been disadvantages great interest of in natu- ralmimicking enzymes therelated proven to highhigh-cost substrate preparation specificity an andd superiorpurification, catalytic low activities. stability, Moreover, and difficul- tiesthe in development storage and ofreuse synthetic [1,3]. approaches Thus, allied could with overcome advances the in disadvantages nano- and biotechnology, of natural enzymes related to high-cost preparation and purification, low stability, and difficulties in artificial enzyme mimics have been extensively studied [7,9]. storage and reuse [1,3]. Thus, allied with advances in nano- and biotechnology, artificial enzymeThe term mimics “nanozyme” have been extensively appeared studiedfor the [ 7firs,9].t time in a work by Scrimin, Pasquato, and co-workersThe term “nanozyme”in 2004 to describe appeared the for excellent the first time catalytic in a work properties by Scrimin, of Pasquato,a multivalent and sys- temco-workers comprising in 2004ligand-functionalized to describe the excellent thiols catalytic on Au nanoparticles properties of a (NPs) multivalent [10]. Since system the no- tablecomprising discovery ligand-functionalized of the intrinsic catalytic thiols on activity Au nanoparticles of magnetic (NPs) Fe [310O].4 NPs Since as the peroxidase notable in 2007,discovery nanozymes of the intrinsicrefer to catalyticnanomaterials activity with of magnetic enzyme-like Fe3O4 NPscharacteristics as peroxidase [5,9]. in 2007, The field hasnanozymes been expanding refer to nanomaterials quickly and with nanomaterials enzyme-like characteristicswith enzyme-mimicking [5,9]. The field activities, has been like expanding quickly and nanomaterials with enzyme-mimicking activities, like Au NPs, Au NPs, Fe3O4 NPs, fullerene derivatives, among many others, have attracted great inter- Fe O NPs, fullerene derivatives, among many others, have attracted great interest and led est and3 4 led to important benchmarks [9,11] (Figure 1). to important benchmarks [9,11] (Figure1).

Figure 1. Figure 1. TimelineTimeline highlighting highlighting relevant relevant histor historicalicalbenchmarks benchmarks of nanozymeof nanozyme development. development. (Reproduced (Reproduced with permission with permission [9], [9], CopyrightCopyright 2019, 2019, The RoyalRoyal Society Society of of Chemistry). Chemistry). Nanomaterials as enzyme mimics present many advantages in comparison with nat- uralNanomaterials enzymes. Either as asenzyme single ormimics multi-components present many like advantages composites in or comparison doped materials, with nat- uralnanozymes enzymes. have Either high as surface-to-volumesingle or multi-components ratio, tunable like catalytic composites activity, or and doped plenty materials, of nanozymessurface reactive have specieshigh surface-to [4,12]. These-volume features ratio, are combined tunable with catalytic cheaper activity, and simpler and man-plenty of surfaceufacturing reactive processes, species long-term [4,12]. stability,These features and robustness are combined to harsh environments.with cheaper However, and simpler some other traits are not so favorable, such as toxicity in biological systems and lack of selectivity, and the catalytic activities of most nanozymes is still low, which may limit the Biosensors 2021, 11, x FOR PEER REVIEW 3 of 19

Biosensors 2021, 11, 152 3 of 19 manufacturing processes, long-term stability, and robustness to harsh environments. However, some other traits are not so favorable, such as toxicity in biological systems and lack of selectivity, and the catalytic activities of most nanozymes is still low, which may limitrange the of range applications. of applications. Thus, nanozymes Thus, nanozymes as artiﬁcial as artificial catalysts catalysts are a likely are a choice, likely choice, but the butﬁeld the still field faces still many faces challengesmany challenges (Figure (Figure2). 2).

FigureFigure 2. 2. ComparisonComparison of of some some characteristics characteristics be betweentween nanozymes nanozymes and and natural natural enzymes. enzymes.

2.1.2.1. Classification Classification and and Enzyme-Like Enzyme-Like Activities Activities RegardingRegarding artificial enzymeenzyme mimics, mimics, several several nanomaterials nanomaterials with with inherent inherent catalytic catalytic activity ac- tivityhave have been studiedbeen studied as highly as highly stable stable and low-cost and low-cost approaches. approaches. These These nanomaterials nanomaterials can be cancategorized be categorized in main in groupsmain groups regarding regarding their composition,their composition, namely namely into metal, into metal, metal metal oxide, oxide,carbon-based carbon-based materials, materials, and their and hybrids their hybr [13].ids A [13]. variety A variety of nanomaterials of nanomaterials has been has studied been as artificial enzyme mimics, including CeO NPs, Fe O NPs, Pt nanomaterials, Au NPs, studied as artificial enzyme mimics, including2 CeO2 3NPs,4 Fe3O4 NPs, Pt nanomaterials, Aubimetal NPs, andbimetal trimetal and trimetal NPs, graphene NPs, graphene oxide, carbon oxide, nanotubes, carbon nanotubes, fullerene fullerene derivates, derivates, quantum dots, metal organic frameworks (MOFs) as some common examples [3,7,14–16] (Figure3). quantum dots, metal organic frameworks (MOFs) as some common examples [3,7,14–16] The discovered nanozymes so far can broadly function as oxidoreductases, namely as (Figure 3). peroxidase, oxidase, superoxide dismutase (SOD), and catalase mimics [9]. Other enzyme- The discovered nanozymes so far can broadly function as oxidoreductases, namely like activities by carbon-based materials, Zr- and Cu-based MOFs and Au NPs modified as peroxidase, oxidase, superoxide dismutase (SOD), and catalase mimics [9]. Other en- with catalytic monolayers have also been reported, namely, hydrolase activity, which zyme-like activities by carbon-based materials, Zr- and Cu-based MOFs and Au NPs mod- catalyzes chemical bond hydrolysis (e.g., nuclease, esterase, phosphatase, protease, and ified with catalytic monolayers have also been reported, namely, hydrolase activity, which silicatein) [7,9]. Laccase-like activity has been demonstrated in Cu-based MOFs based on catalyzes chemical bond hydrolysis (e.g., nuclease, esterase, phosphatase, protease, and guanosine monophosphate coordinated copper [17]. Multi-nanozymes where two or more silicatein) [7,9]. Laccase-like activity has been demonstrated in Cu-based MOFs based on types of enzymes are mimicked can also be observed in some nanomaterials like Au, Pt, guanosine monophosphate coordinated copper [17]. Multi-nanozymes where two or and CeO2 [9,18,19]. These capabilities can be expanded by using bimetallic NPs that can more types of enzymes are mimicked can also be observed in some nanomaterials like Au, achieve not only single- but also multiple-enzyme mimicking, or nanocomposites to benefit Pt,from and their CeO synergistic2 [9,18,19]. effectsThese andcapabilities enhanced can catalytic be expanded performance by using [20 ,bimetallic21] (Figure NPs3). that can achieve not only single- but also multiple-enzyme mimicking, or nanocomposites to benefit from their synergistic effects and enhanced catalytic performance [20,21] (Figure 3). Peroxidases, such as HRP, are widely used in biosensor devices and they catalyze the oxidation of substrates by a peroxide, such as H2O2. After finding the novel properties of Fe3O4 magnetic NPs, their peroxidase-like activity was used in the detection of H2O2 and Biosensors 2021, 11, x FOR PEER REVIEW 4 of 19

glucose [22]. The nanozyme catalyzed the oxidation of the substrate 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS) by H2O2 to the oxidized colored product. Moreover, by combining this reaction with the catalytic oxidation of glucose by using GOx, it was possible to develop a colorimetric assay for glucose detection [22]. Since then, the field has evolved rapidly and metals, metal oxides, MOFs and carbon- based nanomaterials have been studied as peroxidase-like mimics [23,24]. In a work by Cui et al., 2015, a simple colorimetric biosensing platform was proposed based on growing Prussian blue (PB) on the microporous MOF MIL-101(Fe), forming uniform octahedral nanostructures with highly efficient catalytic activity for H2O2 [23]. PB crystals are made of iron ions coordinated by CN bridges, possess a high surface area, also exhibiting intrinsic high peroxidase-like activity. Moreover, the outer surfaces of these PB/MIL-101(Fe) nanostructures were successfully modified, rendering them biocompatible while maintaining their activity [23] (Figure 4). Another interesting example was the application of a peroxidase-like activity to successfully detect thrombin in plasma by preparing a fibrinogen-modified bismuth-gold (Fib-Bi-Au) NPs [25]. The Fib-Bi-Au NPs catalyzed the oxidation of Amplex Red in the presence of H2O2, and this simple fluorescence-based assay enabled detecting thrombin with limit of detection of 2.5 pmol L−1 and revealing a promising clinical application [25] (Figure 5A). The majority of nanozymes applied in detection exhibit peroxidase-like activity; thus, numerous examples are found in the literature with a broad range of nanomaterials [7,26]. Recently, a covalent organic framework (COF) nanozyme has been developed by incorporating an iron porphyrin unit in the COF backbone as the active center and L-histidine as the substrate binding site for selective chiral Biosensors 2021, 11, 152 recognition. The peroxidase-like activity was shown to be enantioselective and 4with of 19 higher activity than the natural HRP, while both the activity and selectivity can be easily modulated by changing the doped amino acids and their content [27].

FigureFigure 3. 3. SchematicSchematic illustration illustration of of some some nano nanomaterialsmaterials mimicking mimicking enzymes, enzymes, the the most most studied studied enzyme-like enzyme-like activities, activities, and and commoncommon ligands ligands to to tune tune the the catalytic catalytic efficiency efﬁciency and and selectivity of nanozymes.

Peroxidases, such as HRP, are widely used in biosensor devices and they catalyze the oxidation of substrates by a peroxide, such as H2O2. After finding the novel properties of Fe3O4 magnetic NPs, their peroxidase-like activity was used in the detection of H2O2 and glucose [22]. The nanozyme catalyzed the oxidation of the substrate 2,20-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS) by H2O2 to the oxidized colored product. Moreover, by combining this reaction with the catalytic oxidation of glucose by using GOx, it was possible to develop a colorimetric assay for glucose detection [22]. Since then, the field has evolved rapidly and metals, metal oxides, MOFs and carbon-based nanomaterials have been studied as peroxidase-like mimics [23,24]. In a work by Cui et al., 2015, a simple colorimetric biosensing platform was proposed based on growing Prussian blue (PB) on the microporous MOF MIL-101(Fe), forming uniform octahedral nanostructures with highly efficient catalytic activity for H2O2 [23]. PB crystals are made of iron ions coordinated by CN bridges, possess a high surface area, also exhibiting intrinsic high peroxidase-like activity. Moreover, the outer surfaces of these PB/MIL-101(Fe) nanostructures were successfully modified, rendering them biocompatible while maintaining their activity [23] (Figure4). Another interesting example was the application of a peroxidase-like activity to successfully detect thrombin in plasma by preparing a fibrinogen-modified bismuth-gold (Fib-Bi-Au) NPs [25]. The Fib-Bi-Au NPs catalyzed the oxidation of Amplex Red in the presence of H2O2, and this simple fluorescence-based assay enabled detecting thrombin with limit of detection of 2.5 pmol L−1 and revealing a promising clinical application [25] (Figure5A). The majority of nanozymes applied in detection exhibit peroxidase-like activity; thus, numerous examples are found in the literature with a broad range of nanomaterials [7,26]. Recently, a covalent organic framework (COF) nanozyme has been developed by incorporating an iron porphyrin unit in the COF backbone as the active center and L-histidine as the substrate binding site for selective chiral recognition. The peroxidase-like activity was shown to be enantioselective and with higher activity than the natural HRP, while both the activity and selectivity can be easily modulated by changing the doped amino acids and their content [27]. BiosensorsBiosensors 20212021, 11,, 11x ,FOR 152 PEER REVIEW 5 of 19 5 of 19

FigureFigure 4. 4.Characterization Characterization by TEM by (a TEM,b) and (a SEM,b) (andc,d) ofSEM microporous (c,d) of MOFmicroporous MIL-101(Fe) MOF as-prepared MIL-101(Fe) as-pre- (pareda,c) and (a after,c) and introducing after introducing Prussian blue, Prussi PB/MIL-101(Fe)an blue, PB/MIL-101(Fe) (b,d), as highly efﬁcient (b,d), peroxidase-like as highly efficient peroxi- mimics,dase-like and mimics, scheme illustratingand scheme the illustrating synthesis of the PB/MIL-101(Fe) synthesis of (e PB/MIL-101(Fe)) (Reproduced with (e permis-) (Reproduced with sion [23] Copyright 2015, The Royal Society of Chemistry). permission [23] Copyright 2015, The Royal Society of Chemistry). Oxidase-like nanozymes are those that catalyze the oxidation of substrates with molecularOxidase-like oxygen. Natural nanozymes enzymes are usuallythose selectivethat cataly for aze given the substrate,oxidation hence of substrates their with mo- nameslecular such oxygen. as GOx. Natural Nanomaterials enzymes such are as Au usually NPs, MnO selective2, or CeO for2 exhibit a given oxidase-like substrate, hence their activity, but they lack selectivity to a given substrate [9,28,29]. Still, oxidase-like nanozymes names such as GOx. Nanomaterials such as Au NPs, MnO2, or CeO2 exhibit oxidase-like are integrated in sensors with proper coupling to biomolecules pertaining to the target. Foractivity, instance, but a colorimetricthey lack sensor selectivity for mercury to ionsa given and DNA substrate molecules [9,28,29]. relied on Still, the oxidase-like oxidase-likenanozymes activity are integrated of bovine serumin sensors albumin-protected with proper silver coupling clusters to (BSA-Ag biomolec NCs).ules pertaining to Thisthe target. activity wasFor stimulatedinstance, bya colorimetric mercury showing sensor high for catalytic mercury activity ions towards and DNA 3,30,5,5 molecules0- relied tetramethylbenzidineon the oxidase-like (TMB), activity in a “switched-on”of bovine serum state [ 30albumin-protected]. Moreover, as mercury silver ions areclusters (BSA-Ag known to bind with two DNA thymine bases, forming base pairs, the sensor could detect DNANCs). molecules. This activity A hairpin was structure stimulated containing by mercurymercury ions showing was disrupted high in catalytic the presence activity towards of3,3 the′,5,5 target′-tetramethylbenzidine DNA, releasing the ions (TMB), that switched in a “switched-on” on the oxidase mimicking state [30]. activity Moreover, of as mercury BSA-Agions are NCs. known The to target bind DNA with could two beDNA detected thymine as low bases, as 10 forming nmol L−1 ,base with pairs, a linear the sensor could −1 rangedetect from DNA 30 tomolecules. 225 nmol L A [hairpin30] (Figure structure5B). In a recent containing work, the mercury oxidase-like ions activity was disrupted in the of Cu/Co bimetallic MOF functionalized with an aptamer was used to detect a protein presence of the target DNA, releasing the ions that switched on the oxidase mimicking on the surface of exosomes [31]. The prepared CuCo2O4 nanorods were surface-modiﬁed −1 withactivity CD63 of aptamers BSA-Ag resulting NCs. The in catalysis target inhibition.DNA could The be aptamers detected disassembled as low as upon10 nmol L , with a exosomelinear range recognition from originating 30 to 225 a nmol recovery L−1 of [30] the oxidase-like(Figure 5B). activity. In a Thisrecent colorimetric work, the oxidase-like methodactivity enabled of Cu/Co the detection bimetallic of exosomes MOF functionalized in a range of 5.6 with× 104 anto 8.9aptamer× 105 particles was used to detect a µL−1, with a detection limit of 4.5 × 103 particles µL−1 [31] (Figure6). protein on the surface of exosomes [31]. The prepared CuCo2O4 nanorods were surface- modified with CD63 aptamers resulting in catalysis inhibition. The aptamers disassembled upon exosome recognition originating a recovery of the oxidase-like activity. This colorimetric method enabled the detection of exosomes in a range of 5.6 × 104 to 8.9 × 105 particles µL−1, with a detection limit of 4.5 × 103 particles µL−1 [31] (Figure 6). Both SOD and catalase enzymes are involved in protecting cells from oxidative dam- age, and likewise to other enzyme mimics, CeO2, Au NPs, among other nanozymes, are artificial alternatives with many potential therapeutic applications [13,32]. Because of their antioxidant properties, many works study the potential benefits of nanozymes with catalase and SOD activities for cell and tissue protection against oxidative stress, in cancer therapy, and as anti-inflammatory and antibacterial agents [33,34]. Biosensors 2021Biosensors, 11, x2021 FOR, 11 PEER, 152 REVIEW 6 of 19 6 of 19

Biosensors 2021, 11, x FOR PEER REVIEW 6 of 19

FigureFigureFigure 5.5. NanozymeNanozyme 5. Nanozyme bioconjugatesbioconjugates bioconjugates for for sensingsensing sensing applications: applications: applications: (A) Scheme(A ()A Scheme) Scheme of bismuth-gold of ofbism bismuth-gold nanoparti-uth-gold nanopar- nanopar- ticlesticles cles preparationpreparation preparation and and peroxidase-likeperoxidase-like peroxidase-like catalyzing catalyzing catalyzing mechanism mechanism mechanism for reaction for for reaction reaction with Amplex with with Amplex Red Amplex (AR) Red (a), Red (AR) (AR) ((aa),), andandand furtherfurther further modiﬁcationmodification with with with ﬁbrinogen fibrinog fibrinog asen probeen as as probe forprobe detection for for detection detection of thrombin of ofthrombin (b thrombin). (Reproduced (b). ( b(Reproduced). with(Reproduced withwith permissionpermission [25 [25]] Copyright CopyrightCopyright 2012, 2012, 2012, The Royal The The Royal Society Royal Society of Society Chemistry); of of Chemistry); Chemistry); (B) Colorimetric (B )( BColorimetric) biosensorColorimetric based biosensor biosensor on oxidase-like activity of bovine serum albumin-protected silver clusters, which is switched on basedbased onon oxidase-likeoxidase-like activityactivity of of bovine bovine serum serum albumin-protected albumin-protected silver silver clusters, clusters, which which is is selectively by mercury ions and applied to detect DNA. (Reproduced with permission [30], Copyright switchedswitched onon selectively byby mercurymercury ions ions and and a ppliedapplied to to detect detect DNA. DNA. (Reproduced (Reproduced with with permis- permis- sion [30],2015, Copyright Elsevier B.V.) 2015, Elsevier B.V.) sion [30], Copyright 2015, Elsevier B.V.)

Figure 6.Figure Nanozyme 6. Nanozyme based basedon CuCo on CuCo2O4 nanorods:2O4 nanorods: SEM SEM characterization characterization (a ()a )and and scheme scheme showingshowing the the modulation modulation of of oxi- Figuredase-like 6. oxidase-likeNanozyme activity of activity basedthe nano of on therods CuCo nanorods by2 Othe4 bynanorods: adsorption the adsorption SEM of the of characterization the aptamer aptamer that that recognizesrecognizes (a) and scheme CD63 CD63 protein showingprotein on exosomes on the exosomes modulation (b), as a(b), asof aoxi- dase-likelabel-free label-freeactivity sensing sensingof approach. the nano approach. (Reprods (Reproducedroduced by the adsorption with with permission permission of the [31], [ 31aptamer], CopyrightCopyright that 2020, recognizes2020, Elsevier Elsevier B.V.). CD63 B.V.). protein on exosomes (b), as a label-free sensing approach. (Reproduced with permission [31], Copyright 2020, Elsevier B.V.). 2.2. Tuning Nanozymes Properties 2.2. TuningThe catalytic Nanozymes activity Properties of nanozymes is related to the physicochemical properties of the nanomaterials,The catalytic activitymainly byof theirnanozymes atomic composition,is related to i.e.,the byphysicochemical atoms present bothproperties in the of theinside nanomaterials, core and on the mainly surface by [34].their Other atomic factor composition,s that affect i.e., the byenzymatic atoms present activity both include in the insidesize, morphology, core and on thesurface surface coating [34]. and Other modification, factors that pH, affect and the temperature enzymatic activity[35,36]. Asinclude a size,result morphology, of their large surfacesurface areacoating that and is easily modification, modified bypH, bioconjugation, and temperature it is possible[35,36]. toAs a tune some properties like size, shape, and composition, leading to final materials with result of their large surface area that is easily modified by bioconjugation, it is possible to improved (or distinct) catalytic activities, robustness to changes in the microenvironment, tune some properties like size, shape, and composition, leading to final materials with improved (or distinct) catalytic activities, robustness to changes in the microenvironment, Biosensors 2021, 11, 152 7 of 19

Both SOD and catalase enzymes are involved in protecting cells from oxidative dam- age, and likewise to other enzyme mimics, CeO2, Au NPs, among other nanozymes, are artificial alternatives with many potential therapeutic applications [13,32]. Because of their antioxidant properties, many works study the potential benefits of nanozymes with catalase and SOD activities for cell and tissue protection against oxidative stress, in cancer therapy, and as anti-inflammatory and antibacterial agents [33,34].

2.2. Tuning Nanozymes Properties The catalytic activity of nanozymes is related to the physicochemical properties of the nanomaterials, mainly by their atomic composition, i.e., by atoms present both in the inside core and on the surface [34]. Other factors that affect the enzymatic activity include size, morphology, surface coating and modification, pH, and temperature [35,36]. As a result of their large surface area that is easily modified by bioconjugation, it is possible to tune some properties like size, shape, and composition, leading to final materials with improved (or distinct) catalytic activities, robustness to changes in the microenvironment, and high stability [8,37]. The fact that some nanomaterials also possess innate magnetic and optical properties, like Fe3O4 and Au NPs, is seen as an additional gain to obtain multiple func- tionalities that are highly valuable for bioaffinity separation or biosensing applications [38]. One of the major drawbacks of nanozymes is the lack of selective recognition of the substrate in contrast to the corresponding natural enzymes. Thus, various methods have been pursued to improve the selectivity of nanozymes. One approach has been to couple peroxidase-like nanozymes with oxidases, producing H2O2 only in the presence of specific substrates [39]. Surface modification with proper ligands or bioreceptors as antibodies, aptamers, and oligonucleotides, through bioconjugation or physisorption, has also been attempted [1] (Figure3). In this context, the combination of nanozymes with MIPs has opened a new avenue to overcome this technological challenge [36]. The area of biomimetic catalysis is vast, and readers are referred to comprehensive literature reviews focusing on types of nanomaterials, classification, catalytic mechanisms, activity, and applications of nanozymes [6,7,9,11,33].

3. Molecular Imprinting Technology Over the past decades, MIPs have proven excellent features in mimicking natural recognition events for a myriad of applications. The most compelling examples concern the replacement of natural antibodies in diagnostic-based immunoassays [40,41]. The molecular imprinting technology has matured to the point of reaching similar or even surpassing the required characteristics in terms of selectivity, robustness, and cost-effective production without involving animal research [42]. Thus, the combination of “plastic” molecular recognition with advances in sensor fabrication has been offering selective and sensitive methods. These devices are promising for low-cost and rapid detection and monitoring of biomarkers of disease (e.g., protein, nucleic acids, metabolites, virus) and compounds of interest in environmental and food analysis [43]. Besides sensors [44], MIPs have several applications, e.g., in drug delivery, tissue engineering, biocatalysis, bioimaging, extraction, among others [45–47]. Given the known advantages of these synthetic materials, research on building catalytic activity into MIPs has for long been considered an exciting area [40].

3.1. MIPs Design The general concept behind the molecular imprinting technology is to build a synthetic polymer receptor via template-guided synthesis. In general, the typical steps for the synthesis of MIP materials can be described as schematically represented in Figure7. This includes the following steps: (1) the template or print molecule is mixed with functional monomers that get positioned spatially around the template and guide the assembly; (2) the polymerization reaction creates a crosslinked matrix around the template; (3) the template is removed from the polymer network leaving a complementary cavity that retains the Biosensors 2021, 11, 152 8 of 19

spatial features, i.e., size and shape, and bonding preferences of the template; (4) the imprinted polymer selectively rebinds the template from complex samples [48]. Different interactions can be established between the template and functional monomers, namely, noncovalent (e.g., hydrogen bonds, ionic interactions, and hydrophobic effects), covalent, and semi-covalent bonding [42,48,49]. Owing to its versatility, the noncovalent approach is the most widely applied method. The best conditions for MIP synthesis can be studied by computational modelling and combinatorial methods [50,51]. Several parameters are relevant to verify the recognition properties of the MIP and its successful application, Biosensors 2021, 11, x FOR PEER REVIEW 8 of 19 namely in sensors. Those include the adsorption capacity, imprinting factor, selectivity factor, and response time [52,53].

FigureFigure 7. Scheme 7. Scheme of of the the principle principle of molecular imprinting imprinting recognition. recognition.

ConcerningConcerning the the polymerization polymerization methods,methods, different different synthetic synthetic routes routes can becan used be used for for MIP preparation. The most widespread are free radical polymerization [54] and sol–gel MIP preparation. The most widespread are free radical polymerization [54] and sol–gel process [55]. In free radical polymerization, it is possible to select among bulk polymer- processization [55] (that. In includes free radical mechanical polymerization, grinding and it is sieving) possible and to more select complex among techniques,bulk polymeri- zationfor example,(that includes to originate mechanical particles grinding (e.g., suspension, and sieving) precipitation, and more complex emulsion, techniques, and seed for example,polymerizations), to originate which particles are also commonly(e.g., susp usedension, [56 ].precipitation, When MIPs are emulsion, combined withand seed polymerizations),electrochemical detection, which are electropolymerization also commonly used is the [56] preferred. When approach MIPs are [44], combined with main with electrochemicaladvantages relating detection, to direct electropolymerization formation of the polymer is the film preferred on the surface approach of the transducer[44], with main advantagesin a wide relating range of to substrates direct formation [57–59] allowing of the thepolymer production film on of boththe surface conductive of the and trans- ducernonconductive in a wide range polymers of substrates [60]. Besides [57–59] the mentioned allowing methodsthe production for MIP of synthesis, both conductive it is also possible to use other strategies for its production, such as surface imprinting, living and nonconductive polymers [60]. Besides the mentioned methods for MIP synthesis, it is polymerization, solid-phase MIP nanoparticles, and various novel technologies that have alsoemerged possible [49 to,61 use]. The other methods strategies chosen for for its MIP production, production such with as desirable surface properties imprinting, and living polymerization,the final configurations solid-phase clearly MIP depend nanoparticles, on the target and moleculevarious novel (e.g., the technologies whole target that or have emergeda small [49,61] fragment. The as methods an epitope, chosen small, for or largeMIP production macromolecules) with anddesirable application, properties also and theconsidering final configurations the costs and clearly simplicity depend of the on processes.the target MIPs molecule is a thriving (e.g., the research whole subject target or a smalland fragment comprehensive as an reviewsepitope, can small, be found or large in the macromolecules) literature covering and methods application, of polymer also con- sideringfabrication the costs and imprinting and simplicity strategies, of the range processe of applications,s. MIPs is and a thriving integration research in biosensing subject and comprehensivedevices [43,46 ,56reviews,62,63]. can be found in the literature covering methods of polymer fab- rication3.2. MIPs and as imprinting Biomimetic Catalystsstrategies, range of applications, and integration in biosensing devices [43,46,56,62,63].The first examples of imprinted polymers as artificial enzymes were reported in the late 1980s [2,41]. The ability to tailor the imprinted sites with functional groups, allied with 3.2.the MIPs robustness as Biomimetic and stability Catalysts of polymer materials, led to considerable research efforts to expandThe first the catalyticexamples applications of imprinted of MIPs. polymers Many differentas artificial approaches enzymes have were been reported explored in the late(e.g., 1980s (non)covalent [2,41]. The imprinting,ability to tailor chemical the impr reactions)inted and sites catalytic with functional MIPs have groups, been prepared allied with theusing robustness analogues and of stability substrates, of transition polymer states, materials, or products led to as considerable templates [64 –research68]. Since efforts the to first important developments, considerable advances in the field have allowed to prepare expand the catalytic applications of MIPs. Many different approaches have been explored more flexible and adaptative structures [2,40]. (e.g., (non)covalentImprinted polymers imprinting, have been chemical combined reactions) with amino and acids, catalytic peptides, MIPs metals, have among been pre- paredothers, using in synergicanalogues strategies of substrates, to realize transition the potential states, of artificialor products enzymes as templates [69–71]. For[64–68]. Sinceexample, the first peroxidase important mimics developments, are useful in theconsi detectionderable of advances the relevant in biomarker the field forhave tumor allowed to prepare more flexible and adaptative structures [2,40]. Imprinted polymers have been combined with amino acids, peptides, metals, among others, in synergic strategies to realize the potential of artificial enzymes [69–71]. For example, peroxidase mimics are useful in the detection of the relevant biomarker for tumor diagnostic 5-hydroxyindole-3-acetic acid (5-HIAA), which is an indoleamine metabolite and is simultaneously a substrate for peroxidase activity [72]. To this end, a multifunctional MIP material was prepared as an artificial peroxidase enzyme. The MIP with hemin as catalytic center and 5-HIAA as template was obtained by fabricating a core-shell magnetic MIP and demonstrated selective oxidation of 5-HIAA. Then, the products of this oxidation were separated and detected by high-performance liquid chromatography (HPLC) to obtain a quantitative detection of 5-HIAA [72]. In biomimetic catalysis, peptide-based enzyme mimicking is also of interest. Major advantages are related to the fact that the building blocks of peptides are amino acids that work as catalytic groups, assemble, and form supramolecular structures through non- Biosensors 2021, 11, 152 9 of 19

diagnostic 5-hydroxyindole-3-acetic acid (5-HIAA), which is an indoleamine metabolite and is simultaneously a substrate for peroxidase activity [72]. To this end, a multifunctional MIP material was prepared as an artificial peroxidase enzyme. The MIP with hemin as catalytic center and 5-HIAA as template was obtained by fabricating a core-shell magnetic MIP and demonstrated selective oxidation of 5-HIAA. Then, the products of this oxidation were separated and detected by high-performance liquid chromatography (HPLC) to obtain a quantitative detection of 5-HIAA [72]. In biomimetic catalysis, peptide-based enzyme mimicking is also of interest. Major advantages are related to the fact that the building blocks of peptides are amino acids that work as catalytic groups, assemble, and form supramolecular structures through noncovalent interactions, all traits of natural enzymes [73]. Based on this approach, many artificial enzymes have been constructed, such as hydrolases, aldolase, oxidoreductase, among others [73–75]. As for other synthetic alternatives, lower catalytic activity and substrate specificity are still challenging, and molecular imprinting can contribute to improving those features. By combining MIPs with peptide assemblies, interesting peptide- based artificial enzymes have been developed. The peptide Fmoc-Phe-Phe-His (Fmoc-FFH) forms stable nanofibers through self-assembly and has the catalytic activity of histidine [75]. Thus, when an MIP for the substrate p-nitrophenyl acetate is prepared on the surface of the catalytic nanofibers, specific binding sites are provided for enhanced artificial hydrolase activity. In addition, the catalytic ability was improved in a wider reaction temperature and pH, and the catalyst was more easily recyclable owing to the introduction of the polymer layer [75]. Another example is the work of Li et al., 2020, who developed a peroxidase- like enzyme by the co-assembly of Hemin and Fmoc-FFH, combined with an imprinted polymer of the substrate ABTS. Since the polymer components can be easily adjusted, the addition of a cationic monomer further enhanced the catalytic activity because the electrostatic interaction created a synergistic effect [73]. DNA oligonucleotides can also have functional properties, such as in molecular recognition and catalysis, the latter known as DNAzymes [76]. Many peroxidase mimics are DNA-based catalysts and oxidize common substrates like TMB and ABTS in the presence of H2O2, which is of interest because colorimetric products enable the development of optical sensors [77]. To improve the selectivity of a peroxidase-mimicking DNAzyme based on a guanine-quadruplex (G4) DNA with a hemin cofactor, an MIP layer for proper substrates was prepared. The DNAzyme incorporated into the MIP exhibited expected selectivity, as well as enhanced stability (DNA degradation was reduced) and activity (higher than that of free DNAzyme) [77,78]. A very promising application of these imprinted DNAzyme nanogels is for biocatalysis inside cells and intracellular therapeutic applications. This material was efficiently internalized, and the MIP was more effective inside cells than the nonimprinted control, leading to the conclusion that the imprinted matrix was effective when located intracellularly [78]. Molecular imprinting technology is extremely versatile as demonstrated by the possibility of imprinting within the nanospace of doubly cross-linked micelles [79,80] (Figure8). In this method, the imprinting is confined within the boundary of surfactant micelles. The obtained MIP NPs have a hydrophobic/hydrophilic core-shell morphology and are water- soluble, making this micellar imprinting very useful for a variety of template molecules [79]. Such imprinted NPs can also be post-functionalized to present catalytic properties. In addition, the catalysis can be fine-tuned owing to the facile modification of size, shape, and depth of the binding pockets. The technology was demonstrated to deploy very efficient artificial phosphodiesterase and esterase-like activities [79,80]. BiosensorsBiosensors 20212021, 11, 11, x, 152FOR PEER REVIEW 10 of 19 10 of 19

FigureFigure 8. 8.Preparation Preparation of catalytic of catalytic molecularly molecularly imprinted imprin nanoparticlested nanoparticles (MINPs) functionalized (MINPs) functionalized with thewith transacylation the transacylation catalyst 4-dimethylaminopyridine catalyst 4-dimethylaminopyridine (DMAP), as a method(DMAP), to molecularlyas a method imprint to molecularly withinimprint cross-linked within cross-lin micelles.ked (Reproduced micelles. with (Reproduced permission [with79], Copyright permission 2017, [79], Wiley-VHCA Copyright AG). 2017, Wiley- VHCA AG). 4. Nanozymes@MIPs Recent advances on tailored MIPs on nanozymes represent a novel avenue for addi- 4. Nanozymes@MIPs tional advances in the field. The synergistic catalysis arising from integrating two different artificialRecent enzymes advances was first on advanced tailored asMIPs a strategy on nanozymes for the formation represent of disulfide a novel bonds avenue for addi- intional peptides advances [81]. In in this the study, field. imprintedThe synergistic polymeric catalysis microzymes arising and from inorganic integrating (Fe3O4 two) different nanozymesartificial enzymes were integrated was first in one advanced process resultingas a strategy in high for product the formation yields and of excellent disulfide bonds in selectivity [81]. peptides [81]. In this study, imprinted polymeric microzymes and inorganic (Fe3O4) Further works expanded the concept based on coating the nanozymes with the MIP layernanozymes containing were binding integrated pockets toin improveone process the selectivity resulting and in thehigh catalytic product performance yields and excellent ofselectivity nanozymes [81] [82.]. Generally, the nanozymes do not present selective recognition, and a specificFurther bioligand works (e.g., antibody,expanded aptamer) the concept is conjugated based on on the coating nanozyme. the Thenanozymes use of such with the MIP ligandslayer containing may compromise binding the highpockets stability to improve and low costthe ofselectivity nanozymes. and Furthermore, the catalytic the performance naturalof nanozymes ligands available [82]. Generally, may not cover the the nanozymes range of emerging do not analytes present [82 selective,83]. These recognition, draw- and a backs can be addressed by growing artificial substrate recognition sites on the nanozymes byspecific molecular bioligand imprinting (e.g., technology. antibody, The aptamer) MIP layer providesis conjugated the selectivity, on the but nanozyme. it has been The use of reportedsuch ligands that the may catalytic compromise activity of the various high stabilit nanozymesy and is alsolow enhanced.cost of nanozymes. Considering Furthermore, nanozymesthe natural as ligands heterogenous available catalysts, may the not substrate cover mustthe range diffuse of to theemerging catalyst surface,analytes and [82,83]. These afterdrawbacks the reaction, can thebe product addressed desorbs, by enablinggrowing enzyme artificial regeneration substrate [84 ].recognition Interestingly, sites on the ifnanozymes the MIP is grown by molecular on the nanozyme, imprinting the several technology. steps of The catalysis MIP could layer be provides enhanced the or selectivity, inhibitedbut it has by been the polymer reported layer. that Thus, the catalytic a surface scienceactivity approach of various supported nanozymes the study is also of enhanced. the three reaction steps to explain the enhanced catalytic activity in the presence of the MIP.Considering This work nanozymes suggested that as theheterogenous substrate concentration catalysts, the near substrate the nanozyme must surfacediffuse to the cata- waslyst enrichedsurface, by and the after imprinted the reaction, polymer withthe pr fasteroduct transportation desorbs, enabling kinetics. enzyme Moreover, regeneration the[84] activation. Interestingly, energy wasif the lower, MIP and is grown the MIP on did th note nanozyme, retain the products, the several facilitating steps of catalysis enzymecould be turnover enhanced [84]. or inhibited by the polymer layer. Thus, a surface science approach supported the study of the three reaction steps to explain the enhanced catalytic activity in the presence of the MIP. This work suggested that the substrate concentration near the nanozyme surface was enriched by the imprinted polymer with faster transportation kinetics. Moreover, the activation energy was lower, and the MIP did not retain the products, facilitating enzyme turnover [84]. For most nanozymes@MIPs, the imprinted polymer is grown to entrap the nanozyme. An example of such strategy has been presented by creating substrate binding Biosensors 2021, 11, x FOR PEER REVIEW 11 of 19 Biosensors 2021, 11, 152 11 of 19

cavitiesFor moston three nanozymes@MIPs, classic nanozymes the imprinted with peroxidase- polymer is grownand oxidase-like to entrap the activities nanozyme. [82] (Fig- Anure example 9). The ofimprinted such strategy nanogels, has been prepared presented by by aqueous creating precipitation substrate binding polymerization, cavities on were threesynthesized classic nanozymes on Fe3O4 nanoparticles with peroxidase- with and peroxidase-l oxidase-likeike activities activity, [82 ]but (Figure also9 on). The CeO2 mim- imprintedicking oxidase nanogels, activity prepared and by Au aqueous NPs mimicking precipitation peroxidases polymerization, [82]. were The synthesized substrate binding onpockets Fe3O4 enablednanoparticles achieving with peroxidase-like remarkable specificity activity, but and also the on CeOenhancement2 mimicking of oxidase the activity of activitynanozymes. and Au In NPsthe mimickingcase of Fe3 peroxidasesO4 and CeO [822, two]. The substrates substrate bindingwere imprinted, pockets enabled namely, TMB achievingand ABTS, remarkable that when specificity oxidized andshow the a enhancementblue and a green of the color, activity respectively. of nanozymes. The Inimprinted the case of Fe O and CeO , two substrates were imprinted, namely, TMB and ABTS, that substrates for3 Au4 NPs were2 TMB and dopamine [82]. The prepared MIP appeared to be when oxidized show a blue and a green color, respectively. The imprinted substrates for Auporous, NPs were allowing TMB and an dopamineefficient substrate [82]. The prepared diffusion. MIP Interestingly, appeared to be and porous, in accordance allowing with ansome efficient reports substrate in the diffusion.literature, Interestingly, surface modifi andcation in accordance of nanozymes with some can reports enhance in thetheir activ- literature,ity [82]. The surface MIPs, modification which were of nanozymesproperly en cangineered enhance with their functional activity [82 charged]. The MIPs, monomers, whichhighly were enhanced properly the engineered selective with substrate functional reco chargedgnition monomers, and the highlycatalytic enhanced activity. the The im- selectiveprovement substrate caused recognition by the MIP and thewas catalytic consistent activity. among The improvementthe various nanozymes caused by the and sub- MIPstrates was analyzed, consistent amongsuggesting the various that nanozyme@M nanozymes andIP substrates may be analyzed,a general suggesting method to that obtain de- nanozyme@MIPsired selectivity may and be activity a general [82] method. to obtain desired selectivity and activity [82].

Figure 9.9. MolecularMolecular imprinting imprinting on on nanozymes: nanozymes: Photographs Photographs and and schemes schemes showing showing bare bare Fe3O Fe4 3O4 nanozyme ( A(A),), with with an an anionic anionic MIP MIP layer layer (T-MIP (T-MIPneg)(negB)) and (B) withand with a cationic a cationic MIP layer MIP (A-MIP layer (A-MIPpos) pos) ((CC)) forfor oxidizing oxidizing two two substrates substrates (TMB (TMB and and ABTS), ABTS), as well as aswell scheme as scheme of imprinting of imprinting the nanogel the (nanogelD). (Reproduced(D). (Reproduced with permission with permission [82], Copyright [82], Copyright 2017, American 2017, ChemicalAmerican Society). Chemical Society).

OtherOther examples examples in in the the literature literature can can be found, be found, such assuch growing as growing a polypyrrole a polypyrrole (PPy) (PPy) based MIP on Fe3O4 nanozymes using methylene blue (MB) as substrate [85]. In this based MIP on Fe3O4 nanozymes using methylene blue (MB) as substrate [85]. In this study, study, the Fe3O4@PPy composite presented superior catalytic properties for MB in the presencethe Fe3O of4@PPy sodium composite persulfate, presented a sulfate radical-basedsuperior cataly oxidant,tic properties in comparison for MB with in the bare presence of sodium persulfate, a sulfate radical-based oxidant, in comparison with bare Fe3O4 Fe3O4 nanozymes. Interestingly, Fe3O4@PPy could still degrade more than 80% of MB afternanozymes. ﬁve recycling Interestingly, cycles [85]. Fe3O4@PPy could still degrade more than 80% of MB after five recyclingThe versatility cycles [85] of imprinted. nanozymes was tested when trying to develop a universal sensorThe for versatility multiplex detection.of imprinted This nanozymes study used was peroxidase-like tested when metal trying (Pt, to Ru, develop and Ir) a univer- nanozymessal sensor for to fabricate multiplex cross-reactive detection. sensor This study arrays toused detect peroxidase-like a variety of analytes metal [ 83(Pt,]. TheRu, and Ir) sensornanozymes arrays to allowed fabricate to discriminate cross-reactive several sensor small arrays biothiol to detect molecules, a variety proteins, of analytes and cells. [83]. The Othersensor successful arrays allowed traits were to discriminate related to the several ability of small identifying biothiol unknown molecules, samples, proteins, as well and cells. as discriminating biothiols in serum and proteins in human urine [83]. Other successful traits were related to the ability of identifying unknown samples, as well as discriminating biothiols in serum and proteins in human urine [83]. The use of Au NPs in the construction of imprinted nanozymes, as a novel method for enhanced selective detection of glucose, was conceived by employing aminophenylboronic acid (APBA) in the MIP shell [86]. The affinity to glucose is assured by the boronic Biosensors 2021, 11, 152 12 of 19 Biosensors 2021, 11, x FOR PEER REVIEW 12 of 19

The use of Au NPs in the construction of imprinted nanozymes, as a novel method for enhancedgroup in selectiveAPBA, which detection can of bind glucose, to adjacent was conceived hydroxyls by employing of saccharides aminophenylboronic under alkaline con- acidditions. (APBA) Moreover, in the MIPto improve shell [86 ].the The catalytic affinity toactivity, glucose heptadecafluoro- is assured by then boronic-octyl bromide groupnanoemulsion in APBA, was which introduced can bind toto adjacentprovide hydroxylsoxygen, which of saccharides resulted under in efficiency alkaline gain of n conditions.about 270-fold Moreover, [86]. These to improve Au NP-based the catalytic GOx activity, mimics heptadecafluoro- are very promising-octyl bromideconsidering the nanoemulsion was introduced to provide oxygen, which resulted in efficiency gain of importance of glucose monitoring. about 270-fold [86]. These Au NP-based GOx mimics are very promising considering the importanceThe oxidase-like of glucose monitoring. activities of other metal nanoparticles have been explored, such as the caseThe oxidase-likeof Au–Pt alloy activities [87]. ofThis other alloy metal was nanoparticles coupled to have magnetic been explored, microspheres such as theto enhance casethe stability of Au–Pt and alloy ensure [87]. This their alloy magnetic was coupled sepa toration. magnetic The microspheres affinity to substrate to enhance was the accom- stabilityplished andby preparing ensure their a magneticMIP containing separation. APBA The affinityas polymer to substrate shells was with accomplished imprinted sites for byglucose. preparing Having a MIP a containingGOx-like activity, APBA as the polymer imprinted shells nanozymes with imprinted had sites about for glucose.200-fold higher Havingcatalytic a GOx-likeefficiency activity, than Au the NPs imprinted [87]. nanozymes had about 200-fold higher catalytic efficiencyRecently, than Au PtPd NPs nanoflowers [87]. (NFs) exhibiting peroxidase-like activity were synthe- Recently, PtPd nanoflowers (NFs) exhibiting peroxidase-like activity were synthesized sized by a surfactant-directing method and further surface-modified with a MIP layer [88]. by a surfactant-directing method and further surface-modified with a MIP layer [88]. The MIPThe wasMIP prepared was prepared by aqueous by aqueous precipitation precipitat polymerizationion polymerization and contained and imprinted contained pock- imprinted etspockets for TMB for (Figure TMB (Figure10). The 10). final The composite final composite (T-MIP-PtPd (T-MIP-PtPd NFs) had better NFs) catalytic had better proper- catalytic ties,properties, achieving achieving a linear range a linear of 0.01–5000 range ofµmol 0.01–5000 L−1 and aµmol detection L−1 and limit a of detection0.005 µmol limit L−1 of 0.005 −1 forµmol colorimetric L for colorimetric detection of detection H2O2. A sensitive of H2O2 colorimetric. A sensitive detection colorimetric of glucose detection was also of glucose possiblewas also through possible a cascadethrough reaction a cascade employing reaction GOx employing [88]. GOx [88].

Figure 10.10.Characterization Characterization of molecularof molecular imprinting imprinting on PtPd on NFsPtPd showing NFs showing spherical spherical nanostructure nanostructure with ﬂower-likeflower-like morphology morphology and and mesoporous mesoporous on theon the surface: surface: SEM SEM images images of PtPdNFs of PtPdNFs (A) and (A) and T- T-MIP-PtPdMIP-PtPd NFs NFs ( (BB);); TEM imagensimagens of of PtPd PtPd NFs NFs (C ()C and) and T-MIP-PtPd T-MIP-PtPd NFs NFs (D). (Reproduced(D). (Reproduced under under the termsterms andand conditionsconditions of of the the Creative Creative Commons Commons Attribution Attribution Non-Commercial Non-Commercial Unported Unported 3.0 3.0 License [ [88],88], Copyright Copyright 2019, 2019, published published by The by The Royal Royal Society Society of Chemistry). of Chemistry).

TheThe research research on on nanocomposites nanocomposites to gain to from gain a synergisticfrom a synergistic effect has beeneffect investigated has been investi- using a combination of PtCu bimetallic NPs and poly(styrene sulfonate) (PSS) function- gated using a combination of PtCu bimetallic NPs and poly(styrene sulfonate) (PSS) func- alized graphene (Gr). Moreover, the surface of PtCu/PSS-Gr was covered by a MIP for detectiontionalized of graphene the ﬂavonoid (Gr). puerarin. Moreover, The the peroxidase-like surface of PtCu/PSS-Gr activity of the was MIP@PtCu/PSS- covered by a MIP for Grdetection was applied of the inflavonoid the colorimetric puerarin. detection The pero ofxidase-like puerarin reaching activity a of limit the ofMIP@PtCu/PSS-Gr detection ofwas1 × applied10−5 mol in Lthe−1 andcolorimetric with a linear detection range of 2pu×erarin10−5 to reaching 6 × 10− 4a mollimit L −of1 [detection89]. As of 1 × proposed10−5 mol L by−1 thisandstudy, with a the linear PSS-Gr range had of good 2 ×dispersity, 10−5 to 6 × stability, 10−4 mol and L−1 a [89]. large As surface proposed area, by this whichstudy, supported the PSS-Gr the had dispersion good dispersity, of PtCu NPs, stabil i.e.,ity, preventing and a large any surface possible area, agglomeration which supported the dispersion of PtCu NPs, i.e., preventing any possible agglomeration that could lead to reduced enzymatic activity. Additionally, as the PtCu/PSS-Gr nanocomposite was covered by the MIP, without the target analyte puerarin, the small H2O2 passes through the polymer and reaches the peroxidase-like enzyme, generating hydroxyl radicals that trigger the oxidation of TMB. However, when puerarin is present and specifically binds the imprinted sites, it acts as barrier to H2O2, leading to decreased catalytic reaction [89]. Biosensors 2021, 11, 152 13 of 19

that could lead to reduced enzymatic activity. Additionally, as the PtCu/PSS-Gr nanocomposite was covered by the MIP, without the target analyte puerarin, the small H2O2 passes through the polymer and reaches the peroxidase-like enzyme, generating hydroxyl radicals that trigger the oxidation of TMB. However, when puerarin is present and specifically binds the imprinted sites, it acts as barrier to H2O2, leading to decreased catalytic reaction [89]. Photooxidase mimics are activated by light for the oxidation of the substrate in the presence of dissolved oxygen [90]. Among the variety of nanomaterials that have been used as photooxidase mimics, the graphite carbon nitride (g-C3N4) is an emerging visible-light-active organic semiconductor with intrinsic fluorescence properties. Hence, applications of g-C3N4 in photocatalysis and photoluminescence-based biosensing are growing [90]. A recent study demonstrated the interesting properties of surface molecular imprinting on g-C3N4 nanozymes for improved detection of L-cysteine in serum [90]. The enzymatic activity was first probed upon blue LED irradiation, leading to oxidation of the chromogenic substrate like TMB without destructive H2O2. Most interestingly, the MIP- g-C3N4 nanozyme, i.e., having TMB imprinted sites on the surface of g-C3N4, showed to suppress the matrix interference from serum samples, enhancing both substrate selectivity and enzyme activity in comparison to bare g-C3N4. Superior properties in terms of enzyme affinity to TMB, in comparison to other inorganic nanozymes, were also suggested in this study [90]. Electrochemiluminescence (ECL) detection has been highly investigated in sensor development. Nonetheless, common ECL reagents have a few disadvantages (e.g., high toxicity, low stability, and environment-sensitive luminescence efficiencies) and aggregation- induced emission (AIE) materials can overcome these limitations in some practical applications [91]. Additionally, nanozyme amplification has been proposed to offer unique advantages to ECL, and an aggregation-induced (AI)-ECL assay combined with Co3O4 nanozymes has been developed for the detection of antibiotic residues, namely, chloramphenicol [91]. In this sensor, the strong and stable signal relied on COF materials with AI-ECL groups (COF-AI-ECL), while the Co3O4 nanozymes worked as the amplification element. The synthesized COF-AI-ECL and Co3O4 were cross-linked to the surface of a gold electrode, followed by the construction of a MIP for selective recognition of chloramphenicol. The sensor showed a detection limit of 1.18 × 10−13 mol L−1, and a linear range of 5 × 10−13 to 4 × 10−10 mol L−1. Each sensor component offered improved sensitivity and selectivity features even when using complex matrix samples, essential to tracing antibiotic residues in food safety control [91]. The possibility of having a sensitive fluorescence system for selective detection of the mycotoxin patulin motivated the development of a system based on Ag NP/flake-like Zn- based MOF nanocomposite (AgNPs@ZnMOF) as an efficient support for MIP [92]. The Ag NPs were created inside the nano-pores of flake-like Zn MOF and the peroxidase-like activity of Ag NPs was greatly improved by the high surface area of MOF while the MIP for patulin ensured the selectivity. The fluorescence intensity, resulting from the product of catalyzed H2O2-terephthalic acid reaction, decreased linearly with increasing concentrations of patulin in a range of 0.1–10 µmol L−1 and a detection limit of 0.06 µmol L−1 [92]. In bioanalytical chemistry, the use of immunoassays is widespread, and the ELISA is considered the gold standard in many applications, owing to the highly sensitive detection [93]. Nonetheless, the research on biomimetic alternatives is very attractive, namely, for reducing production costs and enhancing the reagent stability. Innovation at this level can occur both by using MIPs, also known as synthetic antibodies, and by employing nanozymes as catalytic labels. This biomimetic approach has been proposed for detecting an organophosphate pesticide [93]. In this work, the 96-well array format was accomplished with the synthesis of MIP microspheres by precipitation polymerization, followed by immobilization on the plate by “grafting to” method, assisted by ionic liquid as binder [93]. The nanozyme label relied on the peroxidase-like activity of Pt NPs and was prepared in two steps. First, BSA-hapten conjugates were synthesized and the final Pt@BSA-hapten probe was obtained by mixing with a colloidal solution of Pt NPs. Colori- Biosensors 2021, 11, 152 14 of 19

metric and surface-enhanced Raman scattering were used for signal detection because the 2+ Biosensors 2021, 11, x FOR PEER REVIEWsubstrate TMB was oxidized to a blue TMB , which also possesses a Raman signal.14 Theof 19 proposed method showed a limit of detection of 1 ng mL−1 for triazophos [93]. Various biomimetic alternatives to common ELISA assays have been proposed. The detection of sulfadiazine, an antibacterial compound whose residues in food and environment from concern, was studied using Au@SiO2 and Au@Pt@SiO2 NPs [94,95]. The nanocomposites misuse are of health concern, was studied using Au@SiO2 and Au@Pt@SiO2 NPs [94,95]. Theas labelling nanocomposites markers had as labelling the intrinsic markers peroxida had these-like intrinsic activity peroxidase-like of Au NPs or activity Au@Pt ofbime- Au 2 NPstallic or materials, Au@Pt bimetallic and the SiO materials, NPs offered and the large SiO2 surfaceNPs offered area largeand ease surface of surface area and function- ease of surfacealization. functionalization. This ideal combination This ideal was combination further improved was further by preparing improved MIP by preparingfilms for selec- MIP filmstive recognition for selective and recognition reusability, and allowing reusability, to develop allowing assays to develop whose results assays were whose compara- results wereble to comparablethose obtained to thoseby HPLC obtained [94,95]. by Other HPLC configurations [94,95]. Other ha configurationsve been proposed, have such been as proposed,the use of Pt@SiO such as2 NPs the useandof a MIP Pt@SiO for 2highlyNPsand sensitive a MIP and for selective highly sensitive detection and of histamine selective detectionwith a limit of of histamine detection with of 0.128 a limit mg of L detection−1 [96]. of 0.128 mg L−1 [96]. The multiplicitymultiplicity ofof sensor sensor design design has has been been expanded expanded by by the the report report of aof three-component a three-compo- functionalnent functional cell-mimicking cell-mimicking structure structure bearing bearing a nuclear a nuclear Fe3O4 peroxidase-likeFe3O4 peroxidase-like activity, activity, a shell layera shell of layer a stimuli-responsive of a stimuli-responsive molecularly molecula imprintedrly imprinted hydrogel hydrogel representing representing the cytoplasm, the cy- andtoplasm, a lipid and bilayer a lipid membrane bilayer membrane [97] (Figure [97] 11). (Figure Each component 11). Each component of the tripartite of the system tripartite had uniquesystem functionshad unique to functions deploy an to interesting deploy an switch-likeinteresting colorimetricswitch-like colorimetric system. The system. core Fe 3TheO4 ensurescore Fe3O the4 ensures oxidation the of oxidation the chromogenic of the chromogenic substrate TMB substrate (and similar, TMB like(and ABTS) similar, in like the presenceABTS) in ofthe H 2presenceO2. In turn, of H the2O MIP2. In layerturn, selectivelythe MIP layer recognizes selectively TMB recognizes and is simultaneously TMB and is sensitivesimultaneously to salt concentrationsensitive to salt by concentration swelling or shrinking by swelling [97 ].or Finally,shrinking the [97]. elasticity Finally, of thethe lipidelasticity membrane of the lipid keeps membrane up with the keeps swelling up wi ofth the the anionic swelling MIP of gel the when anionic lowering MIP gel the when salt concentration,lowering the salt until concentration, there is no more until tolerance there is no to more volume tolerance change to and volume the lipid change layer and bursts. the Thus,lipid layer the gradual bursts. Thus, “analog” the gradual gel volume “analog” change gel was volume reflected change in awas “digital” reflected colorimetric in a “dig- outputital” colorimetric because the output burst ofbecause the membrane the burst allows of the access membrane to TMB allows that is access oxidized to TMB to produce that is color.oxidized Interestingly, to produce controlledcolor. Interestingly, access to controlled TMB can alsoaccess be to achieved TMB can by also using be achieved melittin, by a membrane-perturbingusing melittin, a membrane-perturbing amphipathic peptide, amphipathic which introduces peptide, which channels introduces in the membrane. channels Thein the selectivity membrane. is ensured The selectivity by the imprinted is ensured gel, by while the thisimprinted approach gel, enlarges while this the approach potential applicationsenlarges the ofpotential the system applications [97]. of the system [97].

Figure 11. Cell-mimicking protocell of three components with with a a peroxidase-like peroxidase-like iron iron oxide oxide core core as as nucleus, a molecularly imprinted imprinted hydrogel hydrogel shell shell as as cytoplasm, cytoplasm, and and a alipid lipid bilayer bilayer for for biomedical biomedical applications. (Reproduced with permission [[97],97], CopyrightCopyright 2017,2017, AmericanAmerican ChemicalChemical Society).Society).

5. Conclusions and Future Perspectives InIn the quest for cost-effective and robust mimicsmimics of natural enzymes, intense research efforts have achieved achieved progress progress in in improving improving the the selectivity selectivity and and catalytic catalytic efficiency efficiency of ar- of artificialtificial enzymes, enzymes, by by gathering gathering a awide wide range range of of materials materials and and approaches approaches (Figure 1212).). Nanozymes havehave emergedemerged asas the the next next generation generation of of enzyme enzyme mimics mimics to to overcome overcome the the exist- ex- ingisting challenges challenges and and broaden broaden the applicationthe applicatio of syntheticn of synthetic catalysts. catalysts. Despite Despite the demonstrated the demon- advantages,strated advantages, the lack the of lack specificity of specificity has always has always been abeen problem a problem that hindered that hindered a faster a faster and broaderand broader application application of nanozymes. of nanozymes. The The synergy synergy gained gained by usingby using molecular molecular imprinting imprinting technology to create selective substrate binding sites on nanozymes has been reasoned as a solution to solve the obstacle of missing specificity. Simultaneously, MIPs are also biomimetic, low-cost, and stable materials that can be produced on a large scale. The intrinsic catalytic activity of nanozymes can be improved by designing nanomaterial cores Biosensors 2021, 11, 152 15 of 19

technology to create selective substrate binding sites on nanozymes has been reasoned Biosensors 2021, 11, x FOR PEER REVIEWas a solution to solve the obstacle of missing speciﬁcity. Simultaneously, MIPs are15 also of 19

biomimetic, low-cost, and stable materials that can be produced on a large scale. The intrinsic catalytic activity of nanozymes can be improved by designing nanomaterial cores andand properproper surface surface functional functional groups, groups, also also beneﬁting benefiting from from nanocomposites nanocomposites and and doped doped nanomaterials.nanomaterials. Concurrently,Concurrently, thisthis diversitydiversity inin composition,composition, sizes,sizes, shapes,shapes, andand surfacesurface propertiesproperties callscalls for for rigorous rigorous characterization characterization and and standardization standardization of of activity activity if if practical practical implementationimplementation isis foreseen. MIP MIP technology technology is isalso also resourceful, resourceful, contributing contributing with with the pos- the possibility to obtain on-demand tailorable functional moieties and stimuli-responsive sibility to obtain on-demand tailorable functional moieties and stimuli-responsive nanostructures. This biomimetic convergence is a hot research topic and new insights into nanostructures. This biomimetic convergence is a hot research topic and new insights into functional engineered nanozymes@MIPs in the development of biosensing devices are functional engineered nanozymes@MIPs in the development of biosensing devices are ex- expected to ﬁnd auspicious biosensing applications soon. pected to find auspicious biosensing applications soon.

FigureFigure 12. 12.Schematic Schematic representation representation of of the the nanozyme nanozyme action action in in combination combination with with MIP MIP materials. materials.

OnceOnce they they have have surpassed surpassed the the current current limitations limitations and and achieved achieved the enhancedthe enhanced catalytic cata- activitylytic activity and speciﬁcity, and specificity, imprinted imprinted nanozymes nano canzymes be incorporated can be incorporated in portable, in low-cost,portable, andlow- time-savingcost, and time-saving assays for assays novel point-of-carefor novel point- applications.of-care applications. These point-of-care These point-of-care applications ap- linkedplications to low-cost linked to devices low-cost that devices rely on that synthetic rely on materialssynthetic arematerials a huge are opportunity a huge oppor- for developingtunity for developing new tools that new allow tools global that allow efforts global and alliances efforts and in combating alliances in the combating current and the futurecurrent pandemics, and future as pandemics, well as screening as well for as chronicscreening diseases, for chronic allowing diseases, earlier allowing detection earlier and earlierdetection medical and earlier action, medical to improve action treatment, to improve outcomes. treatment outcomes.

AuthorAuthor Contributions: Contributions:Conceptualization, Conceptualization, A.R.C.,A.R.C., M.F.F. andand M.G.F.S.; investigation, A.R.C. A.R.C. and and M.F.F.;M.F.F.; writing—original writing—original draftdraft preparation,preparation, A.R.C.,A.R.C., M.F.F. and V.S.; writing—review and and editing, editing, A.R.C.,A.R.C., M.F.F., M.F.F., V.S., V.S., E.F. E.F. and and M.G.F.S.; M.G.F.S.; supervision, supervision, M.G.F.S. M.G.F.S. and and E.F.; E.F.; project project administration, administration, A.R.C. andA.R.C. M.F.F.; and fundingM.F.F.; funding acquisition, acquisition, A.R.C. A.R.C. and M.G.F.S. and M.G.F.S. All authors All authors have have read read and and agreed agreed to theto publishedthe published version version of the of manuscript. the manuscript. Funding: This research was funded by European Commission, grant number 829040 and National Foundation for Science and Technology, grant number SFRH/BD/130107/2017. Informed Consent Statement: Not applicable Data Availability Statement: Not applicable Biosensors 2021, 11, 152 16 of 19

Funding: This research was funded by European Commission, grant number 829040 and National Foundation for Science and Technology, grant number SFRH/BD/130107/2017. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors gratefully acknowledge funding from the European Commission through the project MindGAP (FET-Open/H2020/GA829040). The author ARC also acknowl- edges funding to National Foundation for Science and Technology, I.P., through the Ph.D. Grant, SFRH/BD/130107/2017. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Tao, X.; Wang, X.; Liu, B.; Liu, J. Conjugation of antibodies and aptamers on nanozymes for developing biosensors. Biosens. Bioelectron. 2020, 168, 112537. [CrossRef] 2. Resmini, M. Molecularly imprinted polymers as biomimetic catalysts. Anal. Bioanal. Chem. 2012, 402, 3021–3026. [CrossRef] [PubMed] 3. Lin, Y.; Ren, J.; Qu, X. Catalytically Active Nanomaterials: A Promising Candidate for Artificial Enzymes. Acc. Chem. Res. 2014, 47, 1097–1105. [CrossRef][PubMed] 4. Wei, H.; Wang, E. Nanomaterials with enzyme-like characteristics (nanozymes): Next-generation artificial enzymes. Chem. Soc. Rev. 2013, 42, 6060–6093. [CrossRef] 5. Gao, L.; Zhuang, J.I.E.; Nie, L.; Zhang, J.; Zhang, Y.U.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2, 577–583. [CrossRef][PubMed] 6. Dong, H.; Fan, Y.; Zhang, W.; Gu, N.; Zhang, Y. Catalytic Mechanisms of Nanozymes and Their Applications in Biomedicine. Bioconjugate Chem. 2019, 30, 1273–1296. [CrossRef] 7. Huang, Y.; Ren, J.; Qu, X. Nanozymes: Classification, Catalytic Mechanisms, Activity Regulation, and Applications. Chem. Rev. 2019, 119, 4357–4412. [CrossRef] 8. Mahmudunnabi, R.G.; Farhana, F.Z.; Kashaninejad, N.; Firoz, S.H.; Shim, Y.-B.; Shiddiky, M.J.A. Nanozyme-based electrochemical biosensors for disease biomarker detection. Analyst 2020, 145, 4398–4420. [CrossRef] 9. Wu, J.; Wang, X.; Wang, Q.; Lou, Z.; Li, S.; Zhu, Y.; Qin, L.; Wei, H. Nanomaterials with enzyme-like characteristics (nanozymes): Next-generation artificial enzymes (II). Chem. Soc. Rev. 2019, 48, 1004–1076. 10. Manea, F.; Houillon, F.B.; Pasquato, L.; Scrimin, P. Nanozymes: Gold-Nanoparticle-Based Transphosphorylation Catalysts. Angew. Chem. Int 2004, 43, 6165–6169. [CrossRef] 11. Liang, M.; Yan, X. Nanozymes: From New Concepts, Mechanisms, and Standards to Applications. Acc. Chem. Res. 2019, 52, 2190–2200. [CrossRef] 12. Jiang, D.; Ni, D.; Rosenkrans, Z.T.; Huang, P.; Yan, X.; Weibo, C. Nanozyme: New horizons for responsive biomedical applications. Chem. Soc. Rev. 2019, 48, 3683–3704. [CrossRef] 13. Lin, Y.; Ren, J.; Qu, X. Nano-Gold as Artificial Enzymes: Hidden Talents. Adv. Mater. 2014, 26, 4200–4217. [CrossRef] 14. Zhang, H.; Lu, L.; Cao, Y.; Du, S.; Cheng, Z.; Zhang, S. Fabrication of catalytically active Au/Pt/Pd trimetallic nanoparticles by rapid injection of NaBH4. Mater. Res. Bull. 2014, 49, 393–398. [CrossRef] 15. Zhang, P.; Sun, D.; Cho, A.; Weon, S.; Lee, S.; Lee, J.; Han, J.W.; Kim, D.-P.; Choi, W. Modified carbon nitride nanozyme as bifunctional glucose oxidase-peroxidase for metal-free bioinspired cascade photocatalysis. Nat. Commun. 2019, 10, 1–14. [CrossRef][PubMed] 16. Li, P.; Wu, C.; Xu, Y.; Cheng, D.; Lu, Q.; Gao, J.; Yang, W.; Zhu, X.; Liu, M.; Li, H.; et al. Group IV nanodots: Newly emerging properties and application in biomarkers sensing. Trends Anal. Chem. 2020, 131, 116007. [CrossRef] 17. Liang, H.; Lin, F.; Zhang, Z.; Liu, B.; Jiang, S.; Yuan, Q.; Liu, J. Multi-copper Laccase Mimicking Nanozymes with Nucleotides as Ligands Multi-copper Laccase Mimicking Nanozymes with Nucleotides as Ligands. ACS Appl. Mater. Interfaces 2016, 9, 1352–1360. [CrossRef] 18. Chen, J.; Wu, W.; Huang, L.; Ma, Q.; Dong, S. Self-Indicative Gold Nanozyme for H2O2 and Glucose Sensing. Chem. Eur. J. 2019, 25, 11940–11944. [CrossRef][PubMed] 19. Liu, X.; Zhou, Y.; Liu, J.; Xia, H. The intrinsic enzyme mimetic activity of platinum oxide for biosensing of glucose, Spec-trochim. Acta Part A Mol. Biomol. Spectrosc. 2021, 248, 119280. [CrossRef] 20. Shin, H.Y.; Park, T.J.; Kim, M. Il Recent Research Trends and Future Prospects in Nanozymes. J. Nanomater. 2015, 2015, 1–11. 21. Liu, X.; Yang, J.; Cheng, J.; Xu, Y.; Chen, W.; Li, Y. Facile preparation of four-in-one nanozyme catalytic platform and the application in selective detection of catechol and hydroquinone. Sens. Actuators B Chem. 2021, 337, 1–9. [CrossRef] 22. Wei, H.; Wang, E. Fe3O4 Magnetic Nanoparticles as Peroxidase Mimetics and Their Applications in H2O2 and Glucose Detection use of the novel properties of Fe3O4 MNPs as peroxidase. Anal. Chem. 2008, 80, 2250–2254. [CrossRef] 23. Cui, F.; Deng, Q.; Sun, L. Prussian blue modified metal–organic framework MIL-101(Fe) with intrinsic peroxidase-like catalytic activity as a colorimetric biosensing platform. RSC Adv. 2015, 5, 98215–98221. [CrossRef] Biosensors 2021, 11, 152 17 of 19

24. Jiang, B.; Duan, D.; Gao, L.; Zhou, M.; Fan, K.; Tang, Y.; Xi, J. Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes. Nat. Protoc. 2019, 13, 1506–1520. [CrossRef][PubMed] 25. Lien, C.; Huang, C.; Chang, H. Peroxidase-mimic bismuth–gold nanoparticles for determining the activity of thrombin and drug screening. Chem. Commun 2012, 48, 7952–7954. [CrossRef] 26. Wang, Q.; Wei, H.; Zhang, Z.; Wang, E.; Dong, S. An emerging alternative to natural enzyme for biosensing and immunoassay. Trends Anal. Chem. 2018, 105, 218–224. [CrossRef] 27. Zhou, Y.; Wei, Y.; Ren, J.; Qu, X. A chiral covalent organic frameworks (COFs) nanozymes with ultrahigh enzymatic activity. Mater. Horiz. 2020, 7, 3291–3297. [CrossRef] 28. Korsvik, C.; Patil, S.; Self, W.T. Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles. Chem. Commun. 2007, 1056–1058. [CrossRef] 29. Pirmohamed, T.; Dowding, J.M.; Singh, S.; Wasserman, B.; Heckert, E.; Karakoti, A.S.; King, J.E.S.; Seal, S.; Self, W.T. Nanoceria exhibit redox state-dependent catalase mimetic activity. Chem. Commun. 2010, 46, 2736–2738. [CrossRef] 30. Wang, G.; Jin, L.; Wu, X.; Dong, Y.; Li, Z. Label-free colorimetric sensor for mercury(II) and DNA on the basis of mercury(II) switched-on the oxidase-mimicking activity of silver nanoclusters. Anal. Chim. Acta 2015, 871, 1–8. [CrossRef][PubMed] 31. Zhang, Y.; Su, Q.; Song, D.; Fan, J.; Xu, Z. Label-free detection of exosomes based on ssDNA-modulated oxidase- mimicking activity of CuCo2O4 nanorods. Anal. Chim. Acta 2021, 1145, 9–16. [CrossRef] 32. He, W.; Zhou, Y.; Wamer, W.G.; Hu, X.; Wu, X.; Zheng, Z.; Boudreau, M.D.; Yin, J. Biomaterials Intrinsic catalytic activity of Au nanoparticles with respect to hydrogen peroxide decomposition and superoxide scavenging. Biomaterials 2013, 34, 765–773. [CrossRef][PubMed] 33. Zhang, Y.; Jin, Y.; Cui, H.; Yan, X.; Fan, K. Nanozyme-based catalytic theranostics. RSC Adv. 2020, 10, 10–20. [CrossRef] 34. Khan, S.; Sharifi, M.; Bloukh, S.H.; Edis, Z.; Siddique, R.; Falahati, M. In vivo guiding inorganic nanozymes for biosensing and therapeutic potential in cancer, inflammation and microbial infections. Talanta 2021, 224, 121805. [CrossRef] 35. Liu, Y.; Wu, H.; Li, M.; Yin, J.; Nie, Z. pH dependent catalytic activities of platinum nanoparticles with respect to the decomposition of hydrogen peroxide and scavenging of superoxide and singlet oxygen. Nanoscale 2014, 6, 11904–11910. [CrossRef][PubMed] 36. Li, Y.; Liu, J. Nanozyme’s catching up: Activity, specificity, reaction conditions and reaction types. Mater. Horizons 2020.[CrossRef] 37. Wang, X.; Hu, Y.; Wei, H. Nanozymes in bionanotechnology: From sensing to therapeutics and beyond. Inorg. Chem. Front. 2016, 3, 41–60. [CrossRef] 38. Li, S.; Zhang, L.; Jiang, Y.; Zhu, S.; Lv, X.; Duan, Z.; Wang, H. In-site encapsulating gold “nanowires” into hemin-coupled protein scaffolds through biomimetic assembly towards the nanocomposites with strong catalysis, electrocatalysis, and fluorescence properties. Nanoscale 2017, 9, 16005–16011. [CrossRef] 39. Menon, S.S.; SooryaValliparambil, C.; Koyappayil, A.; Berchmans, S. Copper-Based Metal-Organic Frameworks as Peroxidase Mimics Leading to Sensitive H2O2 and Glucose Detection. Chem. Sel. 2018, 3, 8319–8324. 40. Ramström, O.; Mosbach, K. Synthesis and catalysis by molecularly imprinted materials. Curr. Opin. Chem. Biol. 1999, 759–764. [CrossRef] 41. Wulff, G. Enzyme-like catalysis by molecularly imprinted polymers. Chem. Rev. 2002, 102, 1–27. [CrossRef] 42. Mosbach, K.; Ramström, O. The emerging tecnhique of Molecular Imprinting and Its Future Impact on Biotechnology. Nat. Biotechnol. 1996, 14, 163–170. [CrossRef] 43. Vaneckova, T.; Bezdekova, J.; Han, G.; Adam, V.; Vaculovicova, M. Application of molecularly imprinted polymers as artificial receptors for imaging. Acta Biomater. 2020, 101, 444–458. [CrossRef] 44. Frasco, M.F.; Truta, L.A.A.N.A.; Sales, M.G.F.; Moreira, F.T.C. Imprinting Technology in Electrochemical Biomimetic Sensors. Sensors 2017, 17, 523. [CrossRef] 45. Haupt, K.; Rangel, P.X.M.; Tse, B.; Bui, S. Molecularly Imprinted Polymers: Antibody Mimics for Bioimaging and Therapy. Chem. Rev. 2020, 120, 9554–9582. [CrossRef] 46. Xu, J.; Miao, H.; Wang, J.; Pan, G. Molecularly Imprinted Synthetic Antibodies: From Chemical Design to Biomedical Applications. Small 2020, 1906644, 1–21. [CrossRef] 47. Silva, M.S.; Tavares, A.P.M.; De Faria, H.D.; Sales, G.F.; Figueiredo, E.C. Critical Reviews in Analytical Chemistry Molecularly Imprinted Solid Phase Extraction Aiding the Analysis of Disease Biomarkers Molecularly Imprinted Solid Phase Extraction Aiding the Analysis of Disease Biomarkers. Crit. Rev. Anal. Chem. 2020, 1–16. [CrossRef] 48. Ye, L.; Mosbach, K. Molecularly imprinted microspheres as antibody binding mimics. React. Funct. Polym. 2001, 48, 149–157. [CrossRef] 49. Dong, C.; Shi, H.; Han, Y.; Yang, Y.; Wang, R.; Men, J. Molecularly imprinted polymers by the surface imprinting technique. Eur. Polym. J. 2021, 145, 110231. [CrossRef] 50. Whitcombe, M.J.; Chianella, I.; Larcombe, L.; Piletsky, S.A.; Noble, J.; Horgan, A. The rational development of molecularly imprinted polymer-based sensors for protein detection. Chem. Soc. Rev. 2011, 40, 1547–1571. [CrossRef] 51. Cowen, T.; Karim, K.; Piletsky, S. Computational approaches in the design of synthetic receptors e A review. Anal. Chim. Acta 2016, 936, 62–74. [CrossRef] 52. Belbruno, J.J. Molecularly Imprinted Polymers. Chem. Rev. 2018, 119, 94–119. [CrossRef] 53. Culver, H.R.; Peppas, N.A. Protein-Imprinted Polymers: The Shape of Things to Come? Chem. Mater. 2017, 29, 5753–5761. [CrossRef][PubMed] Biosensors 2021, 11, 152 18 of 19

54. Salian, V.D.; Byrne, M.E. Living Radical Polymerization and Molecular Imprinting: Improving Polymer Morphology in Imprinted Polymers. Macromol. Mater. Eng. 2013, 298, 379–390. [CrossRef] 55. Moreita, F.T.C.; Moreira-Tavares, A.P.; Sales, M.G.F. Sol-gel-based biosensing applied to medicinal science. Curr. Top. Med. Chem. 2015, 15, 245–255. [CrossRef][PubMed] 56. Chen, L.; Wang, X.; Wenhui Lu, A.; Wu, X.; Li, J. As featured in: Molecular imprinting: Perspectives and applications. Chem. Soc. Rev. 2016, 45, 2137–2211. [CrossRef] 57. Cardoso, A.R.; Marques, A.C.; Santos, L.; Carvalho, A.F.; Costa, F.M.; Martins, R.; Sales, M.G.F.; Fortunato, E. Molecularly- imprinted chloramphenicol sensor with laser-induced graphene electrodes. Biosens. Bioelectron. 2019, 124–125, 167–175. [CrossRef] 58. Tavares, A.P.M.; De Sá, M.H.; Sales, M.G.F. Innovative screen-printed electrodes on cork composite substrates applied to sulfadiazine electrochemical sensing. J. Electroanal. Chem. 2021, 880, 114922. [CrossRef] 59. Martins, G.V.; Marques, A.C.; Fortunato, E.; Sales, M.G.F. Wax-printed paper-based device for direct electrochemical detection of 3-nitrotyrosine. Electrochim. Acta 2018, 284, 60–68. [CrossRef] 60. Crapnell, R.D.; Dempsey-hibbert, N.C.; Peeters, M.; Tridente, A.; Banks, C.E. Talanta Open Molecularly imprinted polymer based electrochemical biosensors: Overcoming the challenges of detecting vital biomarkers and speeding up diagnosis. Talanta Open 2020, 2, 100018. [CrossRef] 61. Piletsky, S.; Canfarotta, F.; Poma, A.; Bossi, A.M.; Piletsky, S. Molecularly Imprinted Polymers for Cell Recognition. Trends Biotechnol. 2020, 38, 368–387. [CrossRef] 62. Bhogal, S.; Kaur, K.; Malik, A.K.; Sonne, C.; Lee, S.S.; Kim, K.H. Core-shell structured molecularly imprinted materials for sens-ing applications. Trends Anal. Chem. 2020, 133, 116043. [CrossRef] 63. El-Schich, Z.; Zhang, Y.; Feith, M.; Beyer, S.; Sternbæk, L.; Ohlsson, L.; Stollenwerk, M.; Wingren, A.G. Review Molecularly imprinted polymers in biological applications. Biotechniques 2020, 69, 407–420. [CrossRef] 64. Leonhardt, A.; Mosbach, K. Enzyme-mimicking polymers exhibiting specific substrate binding and catalytic functions. React. Polym. 1987, 6, 285–290. [CrossRef] 65. Robinson, D.K.; Mosbach, K. Molecular Imprinting of a Transition State Analogue Leads to a Polymer Exhibiting Esterolytic Activity. J. Chem. Soc. Chem. Commun. 1989, 969–970. [CrossRef] 66. Sellergren, B.; Karmalkar, R.N.; Shea, K.J. Enantioselective Ester Hydrolysis Catalyzed by Imprinted. J. Org. Chem. 2000, 4009–4027. [CrossRef] 67. Wulff, G.; Junqiu, L. Design of Biomimetic Catalysts by Molecular Imprinting in Synthetic Polymers: The Role of. Acc. Chem. Res. 2012, 45, 239–247. [CrossRef][PubMed] 68. Mathew, D.; Thomas, B.; Devaky, K.S. Geometrical effect of 3D-memory cavity on the imprinting efficiency of transition-state analogue-built artificial hydrolases. Polym. Bull. 2018, 75, 3883–3896. [CrossRef] 69. Mathew, D.; Thomas, B.; Devaky, K.S.; Mathew, D.; Thomas, B.; Transition, K.S.D. Transition state analogue imprinted polymers as artificial amidases for amino acid p -nitroanilides: Morphological effects of polymer network on catalytic efficiency. Artif. Cells Nanomed. Biotechnol. 2018, 46, 1830–1837. [CrossRef][PubMed] 70. Pan, J.; Liu, S.; Jia, H.; Yang, J.; Qin, M.; Zhou, T.; Chen, Z.; Jia, X.; Guo, T. Rapid hydrolysis of nerve agent simulants by molecularly imprinted porous crosslinked polymer incorporating mononuclear zinc (II)—Picolinamine-amidoxime module. J. Catal. 2019, 380, 83–90. [CrossRef] 71. Stepanova, M.; Solomakha, O.; Ten, D.; Tennikova, T.; Korzhikova-Vlakh, E. Flow-Through Macroporous Polymer Monoliths Containing Artificial Catalytic Centers Mimicking Chymotrypsin Active Site. Catalysts 2020, 10, 1395. [CrossRef] 72. Antuña-Jiménez, D.; Blanco-López, M.C.; Miranda-Ordieres, A.J.; Lobo-Castañón, M.J. Artificial enzyme with magnetic properties and peroxidase activity on indoleamine metabolite tumor marker. Polymer 2014, 55, 1113–1119. [CrossRef] 73. Li, J.; Zhu, M.; Wang, M.; Qi, W.; Su, R.; He, Z. Molecularly imprinted peptide-based enzyme mimics with enhanced activity and specificity. Soft Matter 2020, 16, 7033–7039. [CrossRef][PubMed] 74. Mathew, D.; Thomas, B.; Devaky, K.S.; Mathew, D.; Thomas, B.; Devaky, K.S. Design, synthesis and characterization of enzyme- analogue-built polymer catalysts as artificial hydrolases. Artif. Cells Nanomed. Biotechnol. 2019, 47, 1149–1172. [CrossRef] [PubMed] 75. Zhu, M.; Wang, M.; Qi, W.; Su, R.; He, Z. Constructing peptide-based artificial hydrolases with customized selectivity. J. Mater. Chem. B 2019, 7, 3804–3810. [CrossRef] 76. Joyce, G.F. Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem. 2004, 73, 791–836. [CrossRef] 77. Zhang, Z.; Liu, B.; Liu, J. Molecular Imprinting for Substrate Selectivity and Enhanced Activity of Enzyme Mimics. Small 2017, 13, 1–7. [CrossRef] 78. Zhang, Z.; Liu, J. Intracellular delivery of a molecularly imprinted peroxidase mimicking DNAzyme for selective oxidation. Mater. Horizons 2018, 5, 738–744. [CrossRef] 79. Hu, L.; Zhao, Y. Cross-Linked Micelles with Enzyme-Like Active Sites for Biomimetic Hydrolysis of Activated Esters. Helv. Chim. Acta 2017, 100, 1–10. [CrossRef] 80. Hu, L.; Arifuzzaman, M.; Zhao, Y. Controlling Product Inhibition through Substrate-Specific Active Sites in Nanoparticle-Based Phosphodiesterase and Esterase. ACS Catal. 2019, 9, 5019–5024. [CrossRef] 81. Chen, Z.; Sellergren, B.; Shen, X. Synergistic Catalysis by “Polymeric Microzymes and Inorganic Nanozymes”: The 1 + 1 > 2 Effect for Intramolecular Cyclization of Peptides. Front. Chem. 2017, 5, 1–7. [CrossRef][PubMed] Biosensors 2021, 11, 152 19 of 19

82. Zhang, Z.; Zhang, X.; Liu, B.; Liu, J. Molecular Imprinting on Inorganic Nanozymes for Hundred-fold Enzyme Specificity. J. Am. Chem. Soc. 2017, 139, 5412–5419. [CrossRef] 83. Wang, X.; Qin, L.; Zhou, M.; Lou, Z.; Wei, H. Nanozyme Sensor Arrays for Detecting Versatile Analytes from Small Molecules to Proteins and Cells. Anal. Chem. 2018, 90, 11696–11702. [CrossRef][PubMed] 84. Zhang, Z.; Li, Y.; Zhang, X.; Liu, J. Molecularly imprinted nanozymes with faster catalytic activity and better specificity. Nanoscale 2019, 11, 4854–4863. [CrossRef][PubMed] 85. Hu, Y.; Liu, J.; Xing, H.; Zhou, H.; Wu, M. Fabrication and Application of Magnetically Catalytic Imprinting Nanozymes. Chem. Sel. 2020, 5, 8284–8288. [CrossRef] 86. Fan, L.; Lou, D.; Wu, H.; Zhang, X.; Zhu, Y.; Gu, N.; Zhang, Y. A Novel AuNP-Based Glucose Oxidase Mimic with Enhanced Activity and Selectivity Constructed by Molecular Imprinting and O2-Containing Nanoemulsion Embedding. Adv. Mater. Interfaces 2018, 5, 1–9. 87. Lin, F.; Doudou, L.; Haoan, W.; Yan, C.; Ning, G.; Yu, Z. Catalytic Gold-Platinum Alloy Nanoparticles and a Novel Glucose Oxidase Mimic with Enhanced Activity and Selectivity Constructed by Molecular Imprinting. Anal. Methods 2019, 11, 4586–4592. [CrossRef] 88. Fan, C.; Liu, J.; Zhao, H.; Li, L.; Liu, M.; Gao, J.; Ma, L. Molecular imprinting on PtPd nanoflowers for selective recognition and determination of hydrogen peroxide and glucose. RSC Adv. 2019, 9, 33678–33683. [CrossRef] 89. Guo, L.; Zheng, H.; Zhang, C.; Qu, L.; Yu, L. A novel molecularly imprinted sensor based on PtCu bimetallic nanoparticle deposited on PSS functionalized graphene with peroxidase-like activity for selective determination of puerarin. Talanta 2020, 210, 120621. [CrossRef] 90. Wu, Y.; Chen, Q.; Liu, S.; Xiao, H.; Zhang, M.; Zhang, X. Surface molecular imprinting on g-C3N4 photooxidative nanozyme for improved colorimetric biosensing. Chin. Chem. Lett. 2019, 30, 2186–2190. [CrossRef] 91. Li, S.; Ma, X.; Pang, C.; Wang, M.; Yin, G.; Xu, Z.; Li, J.; Luo, J. Biosensors and Bioelectronics Novel chloramphenicol sensor based on aggregation-induced electrochemiluminescence and nanozyme amplification. Biosens. Bioelectron. 2021, 176, 112944. [CrossRef] 92. Bagheri, N.; Khataee, A.; Habibi, B.; Hassanzadeh, J. Mimetic Ag nanoparticle/Zn-based MOF nanocomposite (AgNPs @ ZnMOF) capped with molecularly imprinted polymer for the selective detection of patulin. Talanta 2018, 179, 710–718. [CrossRef] 93. Yan, M.; Chen, G.; She, Y.; Ma, J.; Hong, S.; Shao, Y.; El-aty, A.M.A.; Wang, M.; Wang, S.; Wang, J. Sensitive and Simple Competitive Biomimetic Nanozyme-Linked Immunosorbent Assay for Colorimetric and Surface-Enhanced Raman Scattering Sensing of Triazophos. J. Agric. Food Chem 2019, 67, 9658–9666. [CrossRef] 94. He, J.; Liu, G.; Jiang, M.; Xu, L.; Kong, F.; Xu, Z. Development of novel biomimetic enzyme- linked immunosorbent assay method based on Au@SiO2 nanozyme labelling for the detection of sulfadiazine. Food Agric. Immunol. 2020, 31, 341–351. [CrossRef] 95. He, J.; Zhang, L.; Xu, L.; Kong, F.; Xu, Z. Development of Nanozyme-Labeled Biomimetic Immunoassay for Determination of Sulfadiazine Residue in Foods. Adv. Polym. Technol. 2020, 2020, 1–8. [CrossRef] 96. Wang, X.; Song, X.; Si, L.; Xu, L.; Xu, Z. A novel biomimetic immunoassay method based on Pt nanozyme and molecularly imprinted polymer for the detection of histamine in foods nanozyme and molecularly imprinted polymer for the detection of histamine in foods. Food Agric. Immunol. 2020, 31, 1036–1050. [CrossRef] 97. Zhang, Z.; Liu, Y.; Zhang, X.; Liu, J. A Cell-Mimicking Structure Converting Analog Volume Changes to Digital Colorimetric Output with Molecular Selectivity. Nano Lett. 2017, 17, 7926–7931. [CrossRef]