Surface modification strategies and the functional mechanisms of gold nanozyme in biosensing and bioassay

Surface modification strategies and the functional mechanisms of gold nanozyme in biosensing and bioassay Sanam Garehbaghi a , Amir M. Ashrafi b , Vojt ech Adam b , Luk a s Richtera b , * a Central European Institute of Technology, Brno University of Technology, Purkynova 123, Brno, CZ-612 00, Czech Republic b Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno, CZ-613 00, Czech Republic ARTICLE INFO Keywords: Gold nanozyme Catalytic activity Surface modification Biosensing and bioassay ABSTRACT Gold nanozymes (GNZs) have been widely used in biosensing and bioassay due to their interesting catalytic activities that enable the substitution of natural enzyme. This review explains different catalytic activities of GNZs that can be achieved by applying different modifications to their surface. The role of Gold nanoparticles (GNPs) in mimicking oxidoreductase, helicase, phosphatase were introduced. Moreover, the effect of surface properties and modifications on each catalytic activity was thoroughly discussed. The application of GNZs in biosensing and bioassay was classified in five categories based on the combination of the enzyme like activities and enhancing/ inhibition of the catalytic activities in presence of the target analyte/s that is realized by proper surface modification engineering. These categories include catalytic activity enhancer, reversible catalytic activity inhibitor, binding selectivity enhancer, agglomeration base, and multienzyme like activity, which are explained and exemplified in this review. It also gives examples of those modifications that enable the application of GNZs for in vivo biosensing and bioassays. 1. Introduction Similar to a wide range of nanomaterials, gold based nanomaterials in the form of nanoparticle, nanocluster or single atom are considered as nanozymes (NZs) since they are endowed with innate catalytic activity like natural enzymes [1,2]. In fact, for the first time in 2004, Manea et al. used the term nanozyme for modified gold nanoparticles (GNPs) with transphosphorylation activity [3]. Gold nanozymes (GNZs) are mainly nanoparticles, which have intrinsic oxidoreductase activities such as peroxidase, oxidase, superoxide dismutase (SOD), catalase, and reductase [4]. GNPs also have partial role in mimicking other catalytic activities like helicase [5] and phosphatase [6]. NZs are preferred compared to natural analogues because of their lower production cost, higher recyclability, reusability and stability in a wider range of pH and temperature [7]. Particularly, GNZs are attracting a significant attention because of their multienzyme like activities, excellent stability, biocompatibility and feasible surface modification [8–10]. GNZs catalyze the substrate reaction by lowering the activation energy similar to a cofactor or metal complex in active site of natural enzymes with a metal ion catalysis mechanism. In a metal-ion catalysis, GNZs similar to multivalent metals, transfer electron/s to the substrate, or make conformational changes in substrate by hydrophobic binding [2,11]. Because of their inherent features GNZs are applied in clinical diagnosis, food safety, and environmental biosensing and bioassays by modulating its enzymatic activity. During detection process using GNZs, a substrate undergoes a catalytic reaction and generates an electrochemical, fluorescent, or colorimetric response [4]. The NZs entered in the biosensing field of study in 2007 [12], however the application of GNZs in biosensing was first reported by Jv et al. in 2010 where they used the peroxidase activity of cysteamine (cysa) modified GNZs for detection of H 2 O 2 and glucose in a cascade system along with glucose oxidase [13]. The use of GNZs in biosensing and bioassays is limited due to their poor catalytic activity and substrate selectivity. Since the catalytic reaction occurs on the surface of GNZs, the surface modification has an important impact on modulating GNZs' biomimetic activity. Further, the surface modification of GNZs makes an interaction with target analyte which is the substrate itself or competes with the substrate to interact with the surface of GNZs [14]. Researchers suggested that the rational surface modification approach in application of GNZs in biosensing and bioassays can enhance the target recognition properties or confer them the target triggered catalytic activity to simulate/inhibit the catalytic activity [15]. Inspired from the catalysis mechanisms of natural enzymes, the surface of GNZs can be modified with functional groups, small molecules, or ions to play a synergistic role in enhancing a specific enzyme-like activity * Corresponding author. E-mail address: [email protected] (L. Richtera). Contents lists available at ScienceDirect Materials Today Bio journal homepage: www.journals.elsevier.com/materials-today-bio https://doi.org/10.1016/j.mtbio.2023.100656 Received 6 February 2023; Received in revised form 19 April 2023; Accepted 3 May 2023 Available online 4 May 2023 2590-0064/©2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Materials Today Bio 20 (2023) 100656 [16]. Indeed, the metal atom in the active site of a natural enzyme is further coordinated with amino acids or ligands containing donor/- acceptor groups to enhance the catalytic activity. Moreover, these groups improve the kinetic of catalytic activity via stabilizing transition state by facilitating adsorption of substrate and transferring electron assisted by catalysis, nucleophilic, or electrophile mechanism. Additionally the H þ or OH  provide an acid-base mechanism of catalysis. These mechanisms of catalysis stabilize the transition state and lower the activation energy [11]. Additionally, unlike the natural enzymes, NZs including GNZs lack the substrate specific binding sites that results in their poor substrate selectivity. The binding site in natural enzymes consists of amino acid residues and spatial structures similar to substrate which can selectively bind to a substrate [16,17]. Moreover, applying modifications with affinity toward the substrate, on the surface of GNZs plays a role similar to binding pockets in active site of natural enzyme. These modifications include bioreceptors like antibody, synthetic antibody, and aptamers that enhance the selectivity of sensing by decreasing the chance of side reactions on the surface of GNZs [18,19]. Furthermore, using the modifications that suppress the catalytic activity of GNZs can enable a mechanism of detection where the catalytic activity is restored in the presence of target analyte [15]. Another type of modifications makes the GNZs less stable upon interaction with the target analyte that leads to the agglomeration which subsequently affect the catalytic activity of GNZs. In a further sensing approach, the surface of the GNZs is subjected to different modifications to provide numerous enzyme activities existing simultaneously or interchangeably named as cascade system (CS) or logic gates (LG), respectively [9,15]. This review aims to summarize the application of GNZs in biosensing and bioassays that is categorized based on the type of the surface modification and functionality in sensing mechanism. This surface modifications for biosensing application are classified as catalytic activity enhancer (CAE), reversible catalytic activity inhibitor (RCAI), binding selectivity enhancer (BSE), agglomeration base (AB), and multienzyme like activity (MEA). These strategies are explained and exemplified in detecting various target analytes from vital/toxic molecules, ions, biomolecules, biomarkers, cancer cells, and pathogens for in vivo and in vitro applications. 2. Mechanism of catalytic activity Similar to natural enzymes, the surface of GNPs can be used for the adsorption and activation of catalytic reactions [20]. This is related to the different enzymatic activities that can be provided by the GNZs because of their proficiency in donating/accepting electron to/from substrate, cleaving bond in substrate, and hydrophobic interaction with substrate while their crystallographic surfaces further have part in stabilizing the List of abbreviations AA acrylic acid AB Agglomeration base ABTS 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) AMPS 2-acrylamido-2-methyl-1-propanesulfonic acid AOx alcohol oxidase APBA aminophenylboronic acid Arg arginine ATP adenosine triphosphate GNPs gold nanoparticles BSA bovine serum albumin BSE Binding selectivity enhancer BSPP bis(p-sulfonatophenyl)phenylphosphane dehydrate dipotassium salt E. coli escherichia coli bacteria CAE catalytic activity enhancer CB [7]. cucurbit [7]uril CMF carbon microfibre CS cascade system CTAB cetyltrimethylammonium bromide cyclen 1,4,7,10-tetraazacyclododecane DAP 2,3-diaminophenazine DMAPMA N-(3-(dimethylamino)propyl)methacrylamide dsDNA double stranded DNA EGDMA ethylene glycol dimethacrylate FCC face-centered cubic GMP guanosine triphosphate GNCs gold nanocluster form nanozymes GNRs gold nanorod shape nanozymes GNZs gold nanozymes GO graphene oxide GOx glucose oxidase HCV hepatitis C virus HER2 human epidermal growth factor receptor 2 HHTP 2,3,6,7,10,11-hexahydroxytriphenylene HIV human immunodeficiency virus HRP horseraddish peroxidase K cat turnover rate K M Michaelis-Menten constant leu leucine LP liposome MAA methacrylic acid MBA N,N0-Methylenebisacrylamide MIP molecularly imprinted polymer MOF metal organic framework MSiO 2 mesoporous silica MWCNT multiwall carbon nanotube NADH nicotinamide adenine dinucleotide NIPAAm N-isopropylacrylamide NLISA nanonzyme-linked immunosorbent assay NS nanosheet OPD o-phenylenediamine P.aerug Pseudomonas aeruginosa bacteria PAMAM poly(amidoamine) PB prussian blue PEG polyethylene glycol PNIPAM poly(N-isopropylacrylamide) PVP polyvinylpyrrolidone QD quantum dot RCAI reversible catalytic activity inhibitor rGO reduced graphene oxide SA sodium alginate SOD Superoxide dismutase S.aureus Staphylococcus aureus bacteria SDZ sulfadiazine Ser serine SERS surface-enhanced raman spectroscopy TSAb target analyte specific antibody TSAg specific antigen for target analyte specific antibody TSAp target analyte specific aptamer TSp target analyte specific peptide ssDNA single stranded DNA ssRNA single stranded RNA TACN 1,4,7-triazacyclononane TMB 3,30,5,50-tetramethylbenzidine V max maximal reaction rate S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 2 transition state. GNPs can also have part in other enzyme like activities like helicase and phosphatase. In this section, three mechanisms of catalytic activities in presence of GNPs are discussed and exemplified. 2.1. Oxidoreductase Oxidoreductase is a group of enzymes with catalytic activity that causes the oxidation or reduction of their substrates by accepting or donating electron to the substrate, which are as peroxidase, oxidase, catalase, SOD, and reductase [21]. In this process, GNZs adsorb the substrate to the surface stabilizing the transition state, so that the reaction of transferring substrate (transition analogue) to product becomes kinetically favorable. During the catalytic activity related to peroxidase, oxidase, catalase, SOD, Two covalently bonded oxygen atoms as in H 2 O 2 , O 2 ,orO 2molecules are adsorbed to Au atoms with one of three modes of adsorption, namely end-on, top-bridge-top, and bridge-bridge, on the surface of GNZs as presented in Fig. 1(a) [22]. Then GNZs transfer electron to the substrates [23]. However GNZs mostly have a crystallographic face-centered cubic (FCC) structure [24], where different facets provide different activation energies to the adsorption of two covalently bonded oxygen atoms [25]. Although the change of electron state in Au atom have a critical role in transferring electrons, further a crystallographic structure of the GNZs provides favorable kinetic for adsorption of H 2 O 2 ,O 2 , and O 2molecules. The summary of the oxidoreductase activities is presented schematically in Fig. 1 (c) [26]. Four types of oxidoreductases were chosen for further discussion as follows based on their frequent application in biosensing and bioassays. Fig. 1. Schematic diagrams to present: (a) adsorbing modes of O 2 on the surface of Au atoms as: (i) The end-on, (ii) top-bridge-top, (iii) and bridge-bridge, redrew from Ref. [22], with permission from The Royal Society of Chemistry, the copyright (2012), (b) peroxidase activity in acidic conditions (left) and catalase activity in basic conditions (right), reproduced from Ref. [27] with permission from the Elsevier, copyright (2015), both activities are described for Au(211) (top), Au(110) (middle) and Au(111) (bottom) in the bottom part of the figure, reproduced from Ref. [7] with permission from the Elsevier, copyright (2021), and (c) Review of the oxidoreductase activities, inspired from Ref. [26] The Royal Society of Chemistry, copyright (2019). (d) Oxidase activity for facet Au(211), (e) Oxidase activity for facet Au(110), (f) Oxidase activity for facet Au(111), (g) SOD activity for facet Au(111), reproduced from Ref. [25] with permission from the American Chemical Society, the copyright (2015), Note that in the figures d–g, the energies are measured in eV. These energies have been calculated by density functional theory, which has been in excellent agreement with experimental results. S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 3 2.1.1. Peroxidase In the case of peroxidase enzymatic reactions, two substrates are used, where H 2 O 2 as an acceptor substrate, oxidizes another donor substrate, and H 2 O molecule is produced, as presented in Fig. 1(b-left) [27]. A peroxidase activity with two substrates can occur on the surface of GNZs with a ping-pong mechanism similar to natural enzyme. This type of reaction on GNZs conforms a similar ping-pong mechanism in two steps similar to natural enzymes [28]. In the first step, H 2 O 2 as the first substrate is adsorbed on the surface of GNZs by the top-bridge-top mode. Then, it dissociates to hydroxide (OH  ) and hydroxyl (OH  ) as products of transition state, later deprotonation of OH  , produces H 2 O and an oxygen adatom stabilized on the surface of GNZ. The second substrate is oxidized by donating an electron to the oxygen adatom in the second step [25]. As an energetically favored facet for a peroxidase reaction, the (100) facet has the most stable transition state [22]. Another facet with peroxidase activity is the (211) [29]. For this type of reaction, an acidic environment is needed which protonate the surface of GNZs, with Bronsted-Lowry protons and enhance the transition state stability. GNZs can show peroxidase activity also similar to Fenton mechanism, in which the Au III converts to Au 0 when H 2 O 2 is decomposed to H þ and HO 2 .In the second part of catalytic cycle Au 0 converts to Au III when H 2 O 2 is decomposed to OH  and OH  [30]. 2.1.2. Oxidase In an oxidase enzymatic reaction, dioxygen is used as an electron acceptor, where it is adsorbed on two gold atoms by the top-bridge-top mode [31]. In this reaction, Au atoms transfer electrons to the adsorbed oxygen molecules, to stabilize the transition state consisting of two adatoms. The oxygen adatom attacks the second substrate removing hydrogen bond in substrate. For the oxidase reaction, the least activation energy occurs on the (211) facet (Fig. 1(d)), while the highest activation energy is observed on (110) (Fig. 1(e)) and (111) facets (Fig. 1(f)) [25]. Different hydrocarbons can be oxidized with GNZs such as amines, alcohols, phenols, carbonyls, benzyls, and sugars, etc. where the product can be either H 2 OorH 2 O 2 molecule. One example is the glucose oxidation, when hydrated glucose anions interact with the gold atoms in GNZs, the interaction produces electron rich gold atoms, which in turn attack the molecular oxygen forming Au I/II -O 2 intermediate. This intermediate helps to transfer electrons from glucose to the molecular oxygen producing H 2 O 2 [22,32]. Another example is nicotinamide adenine dinucleotide (NADH) oxidation which is a coenzyme located inside cells. Since it is carrier of protons and electrons, it has hydrogen donor property. GNZs show the oxidase activity towards NADH, where first GNZs' coordinate with nitrogen atom in NADH, that results in adsorption of NADH on GNZs' surface and producing Au–H complex and NAD þ as products. This is followed by removing of hydride from the surface of GNZs by the O 2 molecule to generate either H 2 OorH 2 O 2 [33]. 2.1.3. Catalase In this reaction H 2 O 2 as the only substrate is adsorbed on the surface of Au atom by an end-on mode. The nucleophilic OH  groups attack substrate and deprotonate it to produce HO 2and H þ as products. Later as a second part of reaction, the formed Au-HO 2intermediate reacts with another H 2 O molecule and Au transfers electrons to HO 2and produces O 2 and H 2 O to complete the catalytic cycle. [15,34], as presented in Fig. 1(b-right) [35]. GNZs shows catalase activity at neutral to alkaline pH, in which the surface of Au atoms is hydrated by OH  groups as Bronsted-Lowry base that stabilizes the transition state. 2.1.4. Superoxide dismutase (SOD) SOD is an enzyme which scavenge superoxide radicals inside cell [36, 37]. GNZs with SOD like activity adsorb O 2with an end-on mode with less activation energy on the surface, protonates this radical and produces HO 2. The rearrangement of two Au-HO 2(Fig. 1(g)), forms H 2 O 2 and O 2 [38] as end products of catalytic reaction. A study shows that adsorption energy of GNZs increases for transition state in the order of Au III -O 2-Au III ,Au III -HO 2,Au III –OH, Au III –H. This leads to losing oxygen properties of GNZs from a range of peroxide to hydride in which makes GNZs more notable than other metal nanozymes [4,39]. 2.1.5. Reductase GNZs further possess reductase activity towards substrates like pnitrophenol [40]. This activity is induced by sodium borohydride, and produces p-aminophenol and changes the color from yellow to colorless [41]. As it is presented in Fig. 2(a), in this reaction p-nitrophenol is adsorbed on the surface of GNZs and H atom produced from decomposition of NaBH 4 is adsorbed on GNZs and at the same time the reaction of H atom and p-nitrophenol produces p-aminophenol [42]. 2.2. Helicase Positively charged GNPs with L-cysteine (cys) modification can unzip a dsDNA similar to helicase enzyme, where GNPs bind double stranded DNA (dsDNA) from negatively charged phosphate backbone, weakening hydrogen bond between base pairs, and unzipping the dsDNA. This is carried out by pulling single stranded DNA (ssDNA) around clusters of GNZs making conformational changes and generating two ssDNAs, as presented in Fig. 2(b) [5,43]. However, to complete a helicase reaction GNPs should detach from ssDNA. Another study shows GNPs has less affinity towards ssDNA/RNA than dsDNA-RNA [44]. 2.3. Phosphatase Phosphatase-like activity is categorized in phosphomonoesterase and phosphodiesterase [45]. Modification of GNPs with organic-ions complex compounds like 1,4,7,10-tetraazacyclododecane (cyclen), and 1,4, 7-triazacyclononane (TACN) provides phosphomonoesterase-like activity, where this modification hydrolyses organic phosphates, by cleaving phosphomonoester bond (Fig. 2(c)). In this catalytic reaction, GNPs play two roles: first, making a hydrophobic interaction with substrate and second, improving the speed of H 2 O dissociation that increases the generation of nucleophilic OH  and H þ [46]. Furthermore, modified GNPs also show phosphodiesterase activity and can cleave phosphodiester bond in RNA. For instance, the dioctylamine has two chiral thiolated heads that can be complexed with two zinc ions on the surface of GNZs. This modification can selectively cleave the uracil dinucleotide as demonstrated in Fig. 2(d) [47]. 3. Surface modification of GNZs for in vitro biosensing mechanisms In this section, five types of biosensing strategies with GNZs due to the modifications are discussed with related examples provided from the literature. Examples describe how interaction of modifying layer and target analyte is considered as a sensing strategy and give information about those biosensing platform properties that improve such as selectivity, sensitivity, linear dynamic range and limit of detection (LOD). One or a combination of these strategies can be applied in biosensors and bioassays according to requirements of detection. More examples are provided in Table 1. 3.1. Catalytic activity enhancer (CAE) Modifying the surface of GNZs with ligands or biomolecules containing nucleophilic, electrophilic functional groups or multivalent elements can enhance the catalytic activity [48]. This is because of stabilizing the transition state via catalysis mechanisms of acid-base, electrophilic, or nucleophilic, that catalyzes the conversion of the substrate to the final products while transferring electron between GNZs and the substrate/s [11]. S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 4 Fig. 2. Schematic diagram to present participation of GNPs in mimicking (a) reductase-like activity [42], reproduced with permission from the Elsevier, copyright (2015), (b) DNA helicase like activity, redrew from Ref. [5], with permission from the American Chemical Society, copyright (2016), (c) phosphatase-like activity, redrew from Ref. [46], with permission from the Elsevier, copyright (2021), (d) and phosphodiesterase like activity, reproduced from Ref. [47], with permission from John Wiley &Sons, the copyright (2016). Table 1 List of Surface modifications and subsequent application of GNZs in biosensing and bioassays. Modification Mechanism Catalytic Activity Target Molecule Linear Range LOD Technique Ref CMF-hemin GOx CAE, CS Peroxidase Glucose 0.1–0.9 mM 0.05 mM Amperometry [95] CMF-hemin AOx CAE, CS Peroxidase Ethanol 0.01–0.15 mM 0.005 mM Amperometry [95] Hemin-MOF-TSAb CAE Peroxidase α -fetoprotein 0.080–43 ng/mL 0.020 ng/ mL Colorimetry [96] Cu-HHTP-MOF NS CAE Peroxidase H 2 O 2 50 nM–16.4 mM 5.6 nM Amperometry [97] Pt@MSiO 2 -TSAg CAE Peroxidase Mumps virus 0.01–100 μ g/mL 10 ng/mL Colorimetry [98] MSiO 2 –Peroxidase, Oxidase E. coli,S.aureus ––Colorimetry [99] PNIPAm CS Oxidase, Peroxidase Glucose 10–70 mM 5.07 mM Colorimetry [100] PNIPAm Peroxidase H 2 O 2 3–15 mM 2.43 mM Colorimetry [100] MoS 2 -QDs CAE Peroxidase Glucose 1–400 μ M 0.068 μ M Colorimetry [101] DNA hydrogel-Ag þ CS Peroxidase Glucose 5–100 μ M 1.7 μ M Fluorescence [102] Zn II -TACN complexe CAE Phosphotase pBR322 plasmid ––Agrose gel electrophoresis [103] Thiolated T10 CAE Peroxidase Hg 2þ 50–2000 nM 10 nM Colorimetry [104] GO CAE Peroxidase H 2 O 2 10 nM–10 mM 1.9 nM Voltammetry [105] GO CAE Peroxidase H 2 O 2 0.01–5mM 2 μ M Colorimetry [105] Tyrosine-TSAp RCAI Peroxidase Norovirus 20–3300 virus/mL 30 virus/ mL Colorimetry [57] Citrate-TSAp RCAI Peroxidase Zearalenone 10–250 ng/mL 10 ng/mL Colorimetry [106] Citrate-TSAp RCAI Catalase Pb 2þ 0.13–53.33 nM 0.07 nM SERS [107] Citrate-TSAp RCAI Peroxidase P.aerug –60 CFU/ mL Amperometry [108] Tyrosine-TSAp RCAI Peroxidase Kanamycin 0.1–60 nM 0.06 nM Voltammetry [109] TSAp RCAI Peroxidase Kanamycin 5–100 nM 2.28 nM Colorimetry [109] Cysa AB Peroxidase Choline ––Colorimetry [110] CTAB-CeO 2 CAE Peroxidase Glucose 0.1–1mM –Colorimetry [111] CTAB-CeO 2 LG Catalase, peroxidase, SOD –– –– [111] CTAB-CeO 2 CAE Peroxidase Glucose 0.1–1mM –Colorimetry [111] (continued on next page) S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 5 Modification of GNZs with biomolecules can enhance catalytic activity. For example, the peroxidase-like activity of GNZs towards glutathione is proved to be enhanced by immobilizing two amino acid residues as active sites on the surface. Selenocysteine is the active site of natural glutathione peroxidase which adsorbs H 2 O 2 . To this end, a synthetic pentapeptide Ser-Arg-Gly-Asp-Cys with SH bonding is self-assembled on the GNZs' surface so that selenium as an active site makes a bonding with the thiol head of glutathione. Fig. 3(a) presents the active site of this modification. A second active site of cysa, was further self-assembled on GNZ to absorb anionic substrate on its amine group. Taking advantage of the two active sites, this structure increases the peroxidase activity on GNZs by 14 times compared to the unmodified GNZs [28]. GNZs in the form of clusters (GNCs) can obtain peroxidase activity upon hybridization with lysozyme. The peroxidase activity is then enhanced by modifying the surface of graphene oxide with as prepared GNZs-lysozyme by electrostatic absorption and later incubation with folic acid. This method can be used to detect cancer cells, because these cells have folate receptors, which helps the cell to absorb the GNZs that enables them to oxidize TMB to generate an analytical signal proportional to the number cancer cells [49]. Apoferritin with hallow structure is composed of six amino acids that contain histidine. Histidine helps to adsorb gold and silver ions inside the cavity of the apoferritin forming Au–Ag-histidine nanozymes. This modification leads to increase the superoxidase and catalase activity of GNZs, because superoxide dismutase enzyme has histidine as the active site, which makes covalent coordination bonding with metallic cofactor, that helps to convert negative charge of protein to positive, and consequently to adsorb superoxide molecule, an example is presented in Fig. 3(b) [50]. When the GNCs-Pt alloy is modified with the guanosine monophosphate (GMP) it has peroxidase activity towards o-phenylenediamine (OPD). The products of this reaction include 2, 3-diaminophenazine (DAP) with a yellow color. The process of production is presented in Fig. 3(c) [51]. During oxidation of glucose with GOx enzyme, H 2 O 2 molecule is produced. Detection of produced H 2 O 2 can be performed by peroxidase activity of GNZs-GMP towards DAP. This colorimetry technique for detection of glucose has a wide linear range of 0.05–0.4 mM and low LOD of 11 μ M. Another form of enhancing the catalytic activity of GNZs is realized when the COOH, or NH 2 on the applied modification interact with the substrate by providing nucleophilic, electrophilic or acid-base catalysis. GNCs in general provide catalase-like activity at alkaline conditions, even Table 1 (continued ) Modification Mechanism Catalytic Activity Target Molecule Linear Range LOD Technique Ref CTAB-CeO 2 LG Catalase, peroxidase, SOD –– –– [111] Au–Pt, APBA monomer, MBA crosslinker BSE Oxidase –– –Colorimetry [79] DMAPMA or AMPS monomer BSE Peroxidase TMB ––Colorimetry [112] AA and NIPAAm monomer, MBA crosslinker CAE Peroxidase ABTS ––Colorimetry [112] Pt–SiO 2 , MAA monomer, EGDMA crosslinker BSE Peroxidase Sulfadiazine ––Colorimetry [80] Graphene-TSAp RCAI Peroxidase Hepatits C Virus ––Colorimetry [113] Graphene-TSAp RCAI Peroxidase Insuline ––Colorimetry [113] Cysa CAE Oxidase Glucose 18–1100 μ M4 μ M Colorimetry [13] Cysa CAE Peroxidase H 2 O 2 2.0–200 μ M 0.5 μ M Colorimetry [13] Ag-hemin-rGO CAE Peroxidase H 2 O 2 10–35 nM 1.26 nM Colorimetry [114] Ag-hemin-rGO CAE Peroxidase Glucose 2–5 μ M 425 nM Colorimetry [114] Pt–SiO 2 CAE Catalase H 2 O 2 0.1 pM–1 mM, 1 mM–10 M 0.1 pM Colorimetry [115] MIL-101 MOFlactate oxidase CS Peroxidase Lactic acid 10–200 μ M 5.0 μ M SER [116] MIL-101 MOF-GOx CS Peroxidase Glucose 10–200 μ M 4.2 μ M SER [116] Histidine CAE Peroxidase Cu 2þ 1–100 nM 0.1 nM Colorimetry [117] Histidine-Cu 2þ RCAI Peroxidase Histidine 0.02–2.0 μ M 20 nM Colorimetry [117] Citrate RCAI Peroxidase Hg 2þ 0.1–200 μ g/mL 1.2 ng/mL Colorimetry [112] BSA CAE Peroxidase Ag þ 0.5–10 μ M 0.204 μ M Colorimetry [118] Fe-MIL-88 MOF-TSAp RCAI Peroxidase HIV virus 30–150 nM 1.4 nM Colorimetry [119] CitrateTSAp RCAI Peroxidase Thrombin 1–100 nM 0.1 nM Colorimetry [120] ssDNA (A15, C15, and T15) LG Peroxidase BSA, HRP 10–1000 nM 10–200 nM 10 nM Colorimetry [121] TSAp RCAI Peroxidase Abrin 0.2–17.5 nM 0.05 nM Colorimetry [122] Graphene, TSAp RCAI Peroxidase Hg 2þ 0.01–0.5 μ M 3.63 nM Colorimetry [123] Graphene, TSAp RCAI Peroxidase Microcystine LR 0.01–1.0 ng/mL 7.14 pg/ mL Colorimetry [123] Cysa, TSp CAE Peroxidase Integrin GPIIb/IIIa 31.25–375 ng/mL –Colorimetry [124] Fe 2 O 3 nanocube, IgG CAE Peroxidase p53-specific autoantibody –0.12 U/mL Colorimetry [125] Fe 2 O 3 nanocube, IgG CAE Peroxidase p53-specific autoantibody –0.08 U/mL Amperometry [125] Heparin AB Peroxidase Heparinase 0.1–3 μ g/mL 0.06 μ g/ mL Colorimetry [126] PEG-Fe 3 O 4 , PEG-folic acid CAE Peroxidase HeLa cell ––Colorimetry [127] GO-SiO 2 -folic acid CAE Peroxidase HeLa cell 250–500,000 cell/mL 250 cell/ mL Colorimetry [128] BSA-GNCs-LPs-TSAb RCAI Peroxidase HER2 6–1000 cell/mL 6 cell/mL Colorimetry [129] TSAb CAE Peroxidase Avian influenza virus 10 pg/mL–10 μ g/mL 1.11 pg/ mL Colorimetry [77] TSAb-Ag shell CAE Peroxidase Norovirus (NS14) 0.001–100 ng/mL 10.8 pg/ mL Colorimetry [130] CTAB, TSAp AB Peroxidase Malachite green 10–500 nM 1.8 nM Colorimetry [131] citrate-casein RCAI Peroxidase Protease 1–0.1 ng/mL 44 ng/mL Colorimetry [132] S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 6 when they are modified with Polyvinylpyrrolidone (PVP) polymers. But, when the GNCs are modified with PAMAM (poly(amidoamine)) polymers, two types of modification can be obtained: The first is when the termination group of PAMAM is OH or COOH, the results show less catalase activity. The second is when termination group is NH 2 , where the modification leads to increase the catalase-like activity at neutral and acidic conditions, and to decrease its SOD and peroxidase activity. This is because the protonated amines of PAMAM in acidic solutions preadsorb OH  on a metal surface, which helps to oxidize H 2 O 2 and produce O 2 [52]. In the case of cetyltrimethylammonium bromide (CTAB) coated GNZs, cys and glutathione with carboxylic modifications enhance the peroxidase activity of GNZs towards TMB. While cys has zwitterionic interaction which further decreases the steric hindrance and increases the peroxidase activity, glutathione has spatial hindrance that leads to less enhancement of the peroxidase activity than cys [35]. A procedure was used to synthesize GNZs with peroxidase activity using reducing agents such as catechol, hydroquinone, resorcinol, vitamin C, pyrogallic acid, sodium citrate, sodium malate, and sodium tartrate. These reducing agents modify the surface of GNZs with negatively charged functional groups. The K M value of these GNZs with a concentration of 0.1 mg/mL, was calculated towards each of H 2 O 2 or TMB substrates. These GNZs with abovementioned modifications have K M value of 0.2489, 0.4971, 0.782, 0.2258 0.8490, 1.634, 2.752, and 2.382 mg/mL towards H 2 O 2 , respectively. While K M values towards TMB as substrate for these GNZs were reported to be 0.0074, 0.0153, 0.0105, 0.003, 0.0059, 0.0026, 0.0008, and 0.0016 mg/mL, respectively. Maximal reaction rate (V max ) of GNZs towards TMB in respect to concentration of GNZs is considered as K cat and were calculated to be as 1.415, 0.464, 0.378, 0.242, 0.219, 0.084, 0.047, and 0.044 IU/mg, respectively [53]. The V max and K M values of GNZs are comparable to kinetic parameters of horseraddish peroxidase (HRP) enzyme as a natural enzyme. These modifications contain different number of functional groups including hydroxyl and carboxyl, that subsequently affect the kinetic parameters of the resulted GNZs. The hydroxyl group plays the role of electron donor and the number of hydroxyl groups determines the electron density distribution. On the other hand, carboxyl groups interact with amine groups of TMB helping in adsorption of TMB as the substrate on the GNZs' surface. In enzymatic activity base biosensors, sensitivity has proportional relation with K M .V max [54] and LOD has proportional relation with K cat /K M , where K cat /K M is considered as catalytic efficiency [55]. Another form of enhancement is based on synergistic effect between GNZs and a second compound, as an example Au III interacts with the nitrogen atom located in a N-heterocyclic ligand, such as benzene and pyridine to form a complex with a catalytic activity. This activity is characterized by the electrostatic interaction and occurs when the nitrogen atom donates a σ -electron to gold and weakly accepting a π -electron [56]. To enhance the GNZs peroxidase activity towards TMB, tyrosine was used to functionalize the GNZs. Since GNZs make coordinate bonding with nitrogen atom in oxidized tyrosine, they achieve 3-fold higher peroxidase activity towards TMB substrate rather than H 2 O 2 that indicates improved substrate selectivity [57]. Coupling GNZs with Prussian blue (PB) enhances the catalytic activity via the synergistic effect of GNZs and PB. Since PB has semiconductive properties, GNZs can excite an electron from the valence to the conductive band of PB, enhancing the peroxidase like activity towards TMB. In this case, GNZs-PB was assembled in a core-shell structure and H 2 O 2 was detected Fig. 3. Schematic diagrams with the CAE sensing mechanism: (a) in GNZs modified with two active sites and its interaction with glutathione, redrew from Ref. [28] with permission from The Royal Society of Chemistry, copyright (2020), (b) to present the synthesis of GNZs-silver-histidine, where functional groups in histidine act as active site with superoxidase activity, reproduced from Ref. [50] with permission from the Elsevier, copyright (2018), (c) GNZs-Pt-GMP modification and its peroxidase activity towards DAP, redrew from Ref. [51] with permission from the Elsevier, copyright (2022), (d) The production of GNZs with Prussian blue modification, reproduced and modified from Ref. [58], with permission from The Royal Society of Chemistry, copyright (2019), and (e) process of detecting cys by the GNZs-heme modification, reproduced from Ref. [64], with permission from The Royal Society of Chemistry, copyright (2021). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 7 electrochemically using an amperometry method without applying any potential as a proof of synergistic action, see Fig. 3(d) [58]. PVP can be used to stabilize GNRs (GNZs in a nanorod shape), this modification can then be remodified with zeolite imidazolate framework-8 shell, which is assembled with Zn II and 2-methylimidazolate and has a pore size of 1–2 nm. The PVP is important here because the C – – O bond in PVP helps for better absorption of Zn II . This combination has synergistic effect in oxidizing two neurotransmitters of serotonin and dopamine, because it has larger adsorption site and higher sensitivity. A previous study [59] used the GNR-PVP zeolite combination for the detection process of serotonin and dopamine. The results were reported in comparison with the combination of GNZs-multiwall carbon nanotube (GNZs-MWCNT). In their study, the electrochemical cyclic voltammetry was used, where in the case of the GNR-PVP-zeolite combination, the limit of detection was found to be increased by 5 times for the serotonin and 7 times for dopamine. A wide linear range as 0.1–25 μ M for serotonin and 0.1–50 μ M for dopamine was observed [59]. To increase the catalytic activity only towards one of the substrates, GNZs must be modified with opposite charge to that of the substrate. Unmodified GNZs show peroxidase-like activity towards both TMB and ABTS (2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) diammonium salt [60]. Peroxidase-like activity of GNZs towards positively charged TMB is enhanced when the GNZs are modified with negatively charged organic compounds such as arabic gum, polyvinylpyrrolidone, or citrate. This catalytic activity of GNZs towards negatively charged ABTS is enhanced, where cysa is used for the modification process that causes a negatively charged surface. Some examples show target analyte competes with the substrate on the surface of GNZs, leading to a decreased catalytic activity as a response in presence of target analyte where substrate has a role in producing a response signal for detection. Nitrite detection is important in food industry since its reaction with amine produces carcinogenic N-nitrosamines. To detect nitrite, the GNCs are modified with histidine since it has peroxidase activity towards TMB and nitrite. In this case, nitrite competes with TMB to be adsorbed on the active sites of the modified GNCs, where increasing the nitrite concentration inhibits the peroxidase activity towards TMB [15]. As another example, GNZs with silica modification have peroxidase activity towards TMB and a stability up to 90 days without any significant changes. However after introducing the dopamine as a neurotransmitter to the measurement solution, the GNZs starts peroxidase activity towards the dopamine because of the electroactive nature of dopamine towards H 2 O 2 , which helps in consuming the H 2 O 2 and fading the blue color of oxidized TMB [61]. In case of cys modified GNCs due to the positive surface charge, ssDNA cannot be adsorbed on the surface of this modification. In the presence of white LED light (30 mW, 30 min), this modification is found to have oxidase activity towards TMB. Thus, when miRNA-155 is used to hybridize with ssDNA, the negatively charged phosphate backbone is exposed and electrostatically adsorbed on the surface of the GNCs-cys modification. The adsorption of the miRNA-155 hybridized ssDNA decreases the catalytic activity towards TMB and is used to detect miRNA-155 concentration [44]. 3.2. Reversible catalytic activity inhibitor (RCAI) Since GNZ act as active site, modifying its surface with some elements or molecules, or ligands makes covalent or electrostatic bonding with the surface of nanozyme and inhibit the catalytic activity of GNZs by passivating its active surface. These modification can be reversible, meaning they recover their catalytic activity in the presence of target analyte [15]. Carboxylic ligands and amine ligands decrease and increase the peroxidase activity of GNZs towards TMB, respectively. Meanwhile each of this ligands has ability to replace the other ligand on the surface of GNZs and can be used as biosensing technique to detect the other ligand [62]. Sulphides and halides make an ionic compound with Au atoms, which is stronger and relatively irreversible in compared to other modifications and can be detected while they decrease the catalytic activity of GNZs [63]. For example, heme is a complex of iron and porphyrin, which has intrinsic peroxidase activity. When GNZs are modified with heme, a coordination bonding from the carboxylic group and metals is created. This modification (GNZs-heme) has oxidase-like catalytic activity characteristics towards TMB because of converting the valance state from Fe III to Fe II . Also, since GNZs-heme hybrid has oxidase-like activity, it is helpful to use heme for the detecting the presence of cys in blood. Because the cys makes thiol bonding with the GNZs, the GNZs-cys bonding is stronger than the GNZs-heme bonding. As a result of this, the cys can replace the heme causing a noticeable decrease in the oxidase activity, and consequently the cys can be detected in the blood (Fig. 3(e)) [64]. The modification of GNZs with adenosine monophosphate, adenosine diphosphate and adenosine triphosphate have peroxidase activity towards TMB. Glutathione detection is possible in this case, as it reacts with GNZs by breaking the modification, creates a stronger thiol bond on GNZs' surface and deters the peroxidase activity [65]. GNCs capped with adenosine 5-monophosphate have peroxidase like activity. GNCs entrapment in a sodium alginate (SA) hydrogel structure cross linked by Zn 2þ as a bivalent ion, makes an egg-box structure. This structure can detect PO3 4ion concentration with a RCAI sensing strategy, because the presence of free PO3 4ions, interacts with the alginates and decompose the hydrogel structure releasing the GNCs from the egg-box structure. For example, phosphate in PBS buffer with 1 mM concentration can be detected within 3 h while it restores GNCs catalytic activity up to 93%, as seen in Fig. 4(a). The results prove that a modified GNCs with a mass ratio of SA to GNCs of 20, has high stability with the least GNCs leakage for 15 days in a pH of 7 in HEPES buffer. Measuring the catalytic activity for modified GNCs with a mass ratio of SA to GNCs of 0.5 shows 85% maintaining stability after two cycles of 300 s and has high stability for 3 days while sustaining the initial catalytic activity [66]. GNZs conjugation with aptamers specific to a type target analyte (TSAp) is a method used to increase the selectivity of sensing towards target analytes [67]. Aptamer with ssDNA or ssRNA structure can make electrostatic interaction or hydrogen bonding with the surface of modified GNZs and reversibly passivate the catalytic activity GNZs towards the substrate. Upon binding to target analyte, the TSAp leaves the surface of GNZs and restore the catalytic activity towards the substrate. The reactivation of the catalytic activity has proportional relation with their concentration [57]. This sensing platform can selectively detect a wide range of target analytes like viruses, bacteria, genome biomarkers, toxins, and other molecules [68]. As an example, acetamiprid (an agricultural pesticide) was sensed using tyrosine modified GNZs. The acetamiprid specific aptamer was denatured and snap chilled and applied to be modified on the surface of GNZs-tyrosine [69]. Glucose oxidase activity of GNZs is used to detect hsa-let-7c miRNA. In this procedure, a ssDNA complementary to hsa-let-7c miRNA type is used to as TSAp to cover the GNZs surface. The target (hsa-let-7c) is a part of miRNAs family that is associated to tumors, and it was possible to sensitively detect it, since it exhibits higher color intensity than all other miRNAs during the experiment [70]. To increase the stability of GNZs in saline environment, the bis(p-sulfonatophenyl)phenylphosphane dihydrate dipotassium salt (BSPP) is used as a capping agent likewise it passivates the glucose oxidase activity of GNZs. However, immobilizing a thiolated guanine rich DNA on GNZs-BSPP recovers its oxidase activity and can be used to reversibly regulate the catalytic activity on the surface of GNZs (see Fig. 4(b)). Another example used DNA modification flexible single strand structure, and the presence of K þ ions make a conformational change producing a compact G-quadruplex shape and blocks the active site of GNZs [71]. Citrate modified GNZs also exhibits peroxidase activity towards amplex red, where oxidation product is a fluorogenic resorufin. In this reaction, the surface of GNZs is reacted and deposited with Bi III elements increasing the sensitivity by 1100 times [72]. This creates a GNZs-Bi modification, which in turn can be modified with fibrinogen to detect thrombin in plasma samples. In this situation, thrombin converts S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 8 fibrinogen to an insoluble fibrin form, which leads to cover the active site of GNZs and decrease the peroxidase activity. Because fibrin passivates part of the GNZs active sites, the un-passivated active sites can still react with the amplex red substrate. This type of inhibitions is referred as a mixed inhibition mechanism [73]. GNZs modified with Pd nanoparticles can lead to the cleavage of allylcarbamate in nonfluorescent pro-rhodamine 110 and produces a fluorescent product. This fluorescent dye is considered as a drug that can be used for therapeutic and fluorescence diagnosis means. To this end, a RCAI modification strategy called biorthogonal is used. In this modification, a ligand with dimethylbenzylammonium as the head is stabilized on the surface of GNZs, that can interact with the cucurbit [7]uril (CB [7]) as a gate keeper. The existence of CB [7] on the surface of GNZs creates a hindrance to inhibit any access of the non-fluorescent pro-- rhodamine 110 substrate to the GNZs [74]. These modified GNZs possess enzyme-like behavior since they have catalytic activity similar to active site of nanozymes and can regulate their catalytic activity in presence of 1-adamantylamine. The 1-adamantylamine is a competing molecule to dimethylbenzylammonium [75]. It can interact with CB [7] and release it from the dimethylbenzylammonium head, recovering the catalytic activity on the surface of GNZs [74]. 3.3. Binding selectivity enhancer (BSE) Antibodies have protein domains which are binding sites with a shape complementary to a target analyte. Conjugation of antibody on GNZs is an approach to address the selectivity inefficiency of GNZs in biosensors and bioassays [76]. A nanonzyme-linked immunosorbent assay (NLISA) was implemented to detect influenza A (H5N1) as a target analyte using GNZs and TSAb (target specific antibody) conjugate. GNZs-TSAb had peroxidase activity towards TMB and high selectivity towards target analyte. As presented in Fig. 4(c), Au III in the chloride salt in aqueous solution is electrostatically interacted with TSAb making an Au III -TSAb mixture. The GNZs-TSAb was synthesized from the mixture, with the addition of TMB as a reducing agent. Influenza virus was adsorbed on wells of immune 96-well plate assay and was quantified based on its interaction with GNZs-TSAb and generation of amplified colorimetric response due to the peroxidase activity of GNZs-TSAb after the second addition of TMB with H 2 O 2 as substrates [77]. Molecularly imprinted polymer (MIP) is a promising approach that can be used in the development of biosensors and bioassays known as synthetic antibodies. This technology causes high sensitivity, high selectivity, and a special molecular memory. MIPs can be synthesized with specific capacity of molecular recognition by introducing recognition properties into synthetic polymers. To prepare MIPs, covalent, noncovalent and sacrificial spacer methods are used to polymerize template molecules with cross-linkers and functional monomers. When the template molecule is removed from the polymer, the target molecules are recognized and adsorbed to the cavities network left behind the removed template molecules (which have the same size, shape, and chemical function selective to the template) [78]. As an example, the GNZs-Pt alloy (with Au:Pt molar ratio of 1:1) was used in preparing of MIPs. Preparation of MIPs included the use of the aminophenylboronic acid (APBA) as the polymer, and the glucose as the template molecules. In the production procedure, the GNZs-Pts are stabilized on the surface of magnetic microspheres. The APBA was then adsorbed on the GNZs-Pt surface through the electrostatic adsorption and the bonds between the nitrogen in the amine group of APBA and gold. The hydroxyl groups in glucose were then interacted with the GNZs-Pt-APBA modification under the oxygen free condition. In the presence of N,N0-methylenebisacrylamide (MBA) as crosslinker, a network structure of APBA was formed as a shell containing the resulted structure. Afterward, to create the MIP cavities, acidic phosphate buffer (pH ¼5) was used to elute the glucose molecules, as demonstrated in Fig. 4(d). In this case, constructing a glucose selective MIP based biosensor was a successful process with 200 fold higher sensitivity than biosensor based on bare GNZs [79]. In another example the sulfadiazine antibiotic (SDZ) was detected in spiked milk and honey media using MIP with antibody mimetic characteristic. The used MIP was prepared using template molecules of SDZ, functional monomers of methacrylic acid, and cross-linkers of EGDMA (ethylene glycol dimethacrylate). Pt–SiO 2 modified GNZs were immobilized in this molecular imprinting. The modified GNZs are more inclined towards adsorbing target molecules and can detect the SDZ with a LOD value of 0.09 mg/L while 50% of catalytic activity was inhibited at 6.1 mg/L concentration of SDZ. when is The colorimetry detection method was used based on inhibiting the GNZs' nanonzymatic activity [80]. Fig. 4. Schematics using the mechanism of RCAI biosensing in the: (a) sodium alginate modified GNCs and illustration of process of releasing the GNCs in presence of phosphate, reproduced from Ref. [66] with permission from the Elsevier, copyright (2021), and (b) glucose oxidase activity of the GNZs-G-quadruplex modification, reproduced from Ref. [71], with permission from the Nature Portfolio, copyright (2015). Schematics using the mechanism of BSE biosensing in the (c) for the detection of influenza virus, redrew from Ref. [77] with permission from the Ivyspring International, copyright (2017), and (d) modification procedure of a GNZs-Pt alloy for MIP base biosensor, reproduced from Ref. [79], with permission from The Royal Society of Chemistry, copyright (2019). S. Garehbaghi et al. Materials Today Bio 20 (2023) 100656 9 Biosens. Bioelectron. 126 (2019) 425–432, https://doi.org/10.1016/ j.bios.2018.10.067. [131] C. Zhao, C.Y. Hong, Z.Z. Lin, X.M. Chen, Z.Y. Huang, Detection of Malachite Green using a colorimetric aptasensor based on the inhibition of the peroxidase-like activity of gold nanoparticles by cetyltrimethylammonium ions, Microchim. 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