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 regard- ing 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 prepa- ration 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 en- zymes) 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 biomedi- cal 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 perspec- tives,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 pro- duction,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