Journal Pre-proof Sensors and biosensors based on metal oxide nanomaterials Biwu Liu, Juewen Liu PII:
To appear in:
Trends in Analytical Chemistry
Received Date: 3 September 2019 Accepted Date: 2 October 2019
Please cite this article as: B. Liu, J. Liu, Sensors and biosensors based on metal oxide nanomaterials, Trends in Analytical Chemistry, https://doi.org/10.1016/j.trac.2019.115690. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Sensors and biosensors based on metal oxide nanomaterials
Biwu Liu, and Juewen Liu*
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada
Email: [email protected]
Abstract Nanomaterials have greatly boosted the biosensor field taking advantage of their optical, catalytic, magnetic and adsorption properties. While noble metal and carbon-based materials have been most often used, metal oxide nanomaterials (MONMs) have recently emerged as a useful platform for their high stability, low cost and versatility in chemical properties. In this article, we first introduce fundamental surface properties of MONMs, especially interactions with DNA. A few sensing strategies are then discussed including the modulation of the catalytic activities of MONMs, and competitive adsorption. DNA can serve as a signaling molecule, and a competitor to probe adsorption of other molecules by MONMs. In addition, DNA can also modulate the catalytic activity of MONMs. Such modulated activities were also observed by several small molecules or ions. In each case, related biosensor designs are introduced. Representative sensors using these mechanisms are reviewed and some future trends are discussed at the end.
Keywords: DNA; aptamers; biointerfaces; nanozymes; adsorption
Abbreviations ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) APTS, aminopropyltriethoxysilane AR, Amplex Red BEGM, Bronchial epithelial cell growth medium BODIPY-ATP, boron dipyrromethene-conjugated adenosine 5'-triphosphate BSA, bovine serum albumin DMEM, Dulbecco's modified eagle medium ELISA, enzyme-linked immunosorbent assay FAM, 6-carboxyfluorescein GO, graphene oxide GOx, glucose oxidase GSH, glutathione HCR, hybridization chain reaction HRP, horse radish peroxidase LOD, limit of detection MES, 2-ethanesulfonic acid MONMs, Metal oxide nanomaterials NTPs, nucleoside triphosphates OPD, o-phenylenediamine OTA, ochratoxin A PAA, poly(acrylic acid) PCA, principle component analysis
PEG, polyethylene glycol PSS, polystyrene sulfonate PZC, point of zero charge SNA, spherical nucleic acid ssDNA, single-stranded DNA TMB, 3,3',5,5'-tetramethylbenzidine UCNPs, upconversion nanoparticles
1. Introduction Sensors and biosensors have significantly improved the quality of life in many aspects such as healthcare, environmental monitoring, and food safety [1-4]. In the past few decades,
developments thanks to the advancements in the material synthesis and characterization . Among various materials, gold and silver [6-9], and carbon-based materials [10-13] have been the most frequently used. For example, the excellent optical [5,14] electric [15,16], and catalytic properties  of gold nanoparticles are very useful for designing biosensors. However, gold is expensive, and its surface is quite susceptible to contamination leading to less reproducible sensor performance. Metal oxides encompass a large number of materials with diverse properties such as catalytic, magnetic properties, UV-absorption, fluorescence quenching, and dielectric properties. For example, TiO2 is a powerful photocatalyst, Fe3O4 is a biocompatible magnetic material useful for separation and drug delivery , while CeO2 in an important oxidative catalyst . These materials are cost-effective and stable, leading to numerous interesting applications. While most of the applications of metal oxides have focused on traditional materials science such as ceramics, optical coating and industry catalysis, interfacing them with biomolecules has been studied as well. In the last decade or so, we have seen many interesting examples of sensors and biosensors involving metal oxides. Metal oxides can serve as a signal transduction element and a target recognition element for sensors. For signal transduction, magnetic, electric and optical based sensing have all been demonstrated. To be focused, this article is limited to optical sensors
involving metal oxide nanomaterials (MONMs). Optical detection can be achieved by visual inspection or highly sensitive fluorescence, both of which can also be quantified with cost-effective detection devices. High quality monodispersed MONMs can be prepared in organic phases with strong ligands (e.g. magnetic iron oxide nanoparticles). However, we are more interested in sensing involving bare oxide surfaces without strong ligands. This can set a basis for subsequent discussion involving a ligand shell. We start by introducing some basic properties of MONMs following by discussing important examples to show the trend of this field in the near future.
2. Surface properties of MONMs Before reviewing the sensor work, we first describe some representative properties of MONMs, which may influence their interactions with biomolecules . We discuss the complexation of various anions, and small molecules, the enzyme-like catalytic activity, and the dissolution of MONMs upon ligand binding. 2.1 Surface charge and ligand exchange The hydration of MONMs involves chemisorption of water, which could dissociate into surface hydroxide  (Figure 1A). Depending the pH, the surface hydroxide can be further pronated/deprotonated, resulting in charged surfaces. In general, raising pH facilitates deprotonation of surface hydroxide, leading to more negatively charged surfaces, while in acidic buffers, the surface hydroxide might be protonated to become positively charged. The pH of transition from positive to negative potential is called point-of-zero-charge (PZC), at which, the overall charge is zero. Table 1 lists the PZC
values of some common MONMs measured by Zhang et al . It needs to be noted that depending on the method of preparation, particle size, the concentration of surface hydroxide, characterization method and buffer conditions, the PZC of the same material may vary a few pH units . At a given pH, different MONMs carry different surface charges. For instance, at physiological pH, most of oxides are negatively charged with only a few exceptions (e.g. ZnO, NiO). Interactions of MONMs with charged biomolecules are inevitable, and such electrostatic interactions often play a critical role in these systems .
Figure 1. Surface properties of MONMs. (A) Chemisorption of water on an oxide surface and its protonation/deprotonation upon pH variation. (B) Different modes of arsenate complexing with iron oxide: a) bidentate binuclear, b) bidentate mononuclear and c) monodentate mononuclear. (C) Binding of dopamine on an oxide surface. (D) Dissolution of MONMs in water, bronchial epithelial cell growth medium (BEGM), and Dulbecco's modified eagle medium (DMEM). (E) A scheme showing that a typical nanozyme with 7
peroxidase-like activity catalyzes the oxidation of chromogenic substrate 3,3′,5,5′tetramethylbenzidine (TMB) in the presence of H2O2. Panel (D) reprinted from ref.  with permission, copyright 2012 American Chemical Society.
Table 1. PZC of MONPs and ζ-potential at pH 7.6. ζ-potential (mV)b MONPs PZCa Al2O3 7.4 -68.3 ± 1.7 CeO2 7.8 -4.2 ± 0.6 CoO 9.4 -21.4 ± 3.62 Cr2O3 9.2 -29.8 ± 0.9 α-Fe2O3 5.3 -29.8 ± 1.2 Fe3O4 7.2 -29.1 ± 3.0 In2O3 8.1 -22.1 ± 2.1 Mn2O3 2.99 -50.0 ± 1.0 NiO 11.4 17.2 ± 0.2 SiO2 1 -38.1 ± 0.8 SnO2 4 -40.5 ± 1.7 TiO2 (anatase) 6.4 -25.8 ± 0.5 WO3 0.3 -49.6 ± 1.5 Y2O3 9.6 -8.8 ± 0.4 ZnO 9.6 23.5 ± 0.7 ZrO2 5.8 -32.3 ± 2.0 a : The PZC values were adapted from reference  with permission, copyright 2012 American Chemical Society; b: The ζ-potential values were from reference  with permission, copyright 2015 American Chemical Society.
In addition to electrostatic interactions, the surface metal species on MONMs can also interact with ligands via coordination. In this regard, the hard-soft-acid-base theory is a useful starting point. In general, metals that can form stable oxides tend to be hard or borderline metals. Some metals such as Ni2+ and Co2+ may very strongly bind to their ligands due to strong coordination interactions and slow ligand exchange kinetics. 8
Although many soft metals can also form oxides such as CuO, HgO, Ag2O, and CdO, we have not seen a lot of sensing applications with them. These metals often form more stable products with sulfur. One concern is the toxicity of these soft thiophilic metals, which also limits their applications. One type of interesting ligands is inorganic anions. For instance, removal of arsenate using magnetic iron oxides has been pursued for decades . Arsenate mainly forms a bidentate binuclear complex with surface iron [26,27], although two other modes are also known (Figure 1B). Chemisorption of sulfate, phosphate, halides, nitrate, carbonate, selenate, and oxalate is well-documented . Besides anions, some biologically important small molecules can also strongly bind on the surface metal sites. For example, dopamine  and catechols  can bind to almost any MONMs with high affinity (Figure 1C). Such small molecules can also serve as a bridge to anchor functional biomolecules (e.g., DNA) on the surface of oxides [30,31]. Finally, the adsorption of biomacromolecules on MONMs may take place via different interaction forces (e.g., electrostatic, coordination, hydrogen bonding). In a later section, we review some fundamental understandings on the adsorption of DNA on MONMs. The interaction of other biomolecules with some typical MONMs was reviewed by Perry and co-workers . 2.2 Stability and reactivity MONMs are often conceived to be very stable, even in biological environments. However, they could dissolve depending on their solubility and ligands. Figure 1D shows the rate of dissolution of various MONMs in water and cell culture medium . ZnO and CuO dissolved significantly in cell culture medium, followed by WO3, NiO, Sb2O3,
CoO. Other oxides are quite stable in terms of metal leaching. However, dissolution may be accelerated by lowering pH, and/or reacting with ligands . For example, dopamine as a ligand can interact with iron oxide, causing Fe leakage and forming a different complex on the surface . Biological thiols (e.g., glutathione, GSH) can reduce and dissolve MnO2 sheets into free Mn2+ . Such dissolution and leaching of metal ions could cause toxicity of these MONMs, while controlled dissolution might also be useful for designing sensors. 2.3 Catalytic properties MONMs have many important properties useful for biosensor development, such as optical properties (ZnO) and magnetic properties (Fe3O4). Recently, efforts have been made to pursue their enzyme-like catalytic activities (Figure 1E), which provided unique opportunities for sensor design. In 2007, the Yan group first reported magnetic Fe3O4 nanoparticles can mimic peroxidase activity and it can oxidize many chromogenic and fluorogenic substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) (ABTS) and in the presence of H2O2 . Following this pioneering work, many other MONMs, such as Fe2O3 , V2O5 , Co3O4 [38,39], and CuO  were reported to mimic peroxidase. Oxidase can use dissolved oxygen to oxidize the substrates in the absence of H2O2, and this is an analytical advantage. In 2009, Asati et al  reported that nanoceria was able to catalyze the oxidation of TMB, ABTS, and dopamine. NiO is also an oxidase mimic but it only reacts with Amplex Red (AR) to produce fluorescent resorufin . Interestingly, CeO2 is not very efficient in catalyzing this reaction. Aside from peroxidase and oxidase, MONMs with catalase , superoxidase , and phosphatase  have
also been demonstrated. More comprehensive reviews on nanozymes have been reported by Qu  and Wei .
3. MONMs in sensor design A typical sensing process involves the recognition of an analyte followed by transducing the event into detectable signals. MONPs can serve in either process or even both. In this section, we describe some representative examples to illustrate the role of MONPs in sensing. 3.1 MONMs for signaling Protein enzymes have been widely used in immunosorbent assays to achieve signal amplification via catalytic turnovers. For instance, in a sandwich-type enzymelinked immunosorbent assay (ELISA), a horse radish peroxidase (HRP) is coupled with an antibody to signal the binding with the antigen. Designs without a capture antibody or a secondary antibody are also possible. Since the enzyme-antibody conjugates are costly to prepare and unstable, it is desirable to replace HRP by more stable nanozymes (Figure 2A). In their seminal work , Yan and co-workers demonstrated such a design (Figure 2B). An antibody (anti-HBV preS1 antibody) was attached on the immobilized antigen (hepatitis B virus surface antigen, preS1). Fe3O4 NPs were functionalized with protein A, which can recognize the bound antibody. The substrates (TMB and H2O2) were introduced to general a colorimetric signal. This assay was successfully used to detect preS1 with a high selectivity against bovine serum albumin (BSA) (Figure 2C).
Figure 2. Examples of MONPs in signaling. (A) Schematic showing a typical design of a sandwich-type ELISA. A MONM replaces the enzyme in this process. (B) A scheme showing the design of an indirect (no capture antibody) ELISA for the detection of antigen preS1. (C) The oxidation of TMB indicated by the absorbance at 652 nm correlated with the concentration of antigen preSA, and BSA was used as a control. (D) Schematic showing a nanoceria-involved ELISA can oxidize ampliflu into stable fluorescent products at neutral pH but not at acidic pH. (E) Images showing the oxidation of ampliflu catalyzed by either nanoceria or HRP with H2O2. Detection of folate receptor expressed in (F) lung carcinoma cells (A-549) and (G) MCF-7 breast carcinoma cells. 12
Panel (B,C) reprinted from ref.  with permission, copyright 2007 Springer Nature Publishing. (D-G) from ref.  with permission, copyright 2011 American Chemical Society.
Nanozymes with oxidase-like activity have also been used in immunoassays without the need of H2O2 and showed unique advantages. Asati et al  used poly(acrylic acid) (PAA) modified nanoceria (PAA-CeO2) as an oxidase mimic, and they found that PAA-CeO2 was able to oxidize AR to a stable fluorescent product resorufin at pH 7.0 (Figure 2D, 2E). However, the resorufin was further oxidized into non-fluorescent resazurin at pH 4, which was the optimal pH for most peroxidase/oxidase-mimics . Interestingly, AR was rapidly oxidized into non-fluorescently products by H2O2 under the catalysis of HRP even at pH 7. Thus, the oxidase-like activity of PAA-CeO2 is pHtunable, which allowed the authors to design ELISAs for biomarkers at neutral pH. To bind the target with higher specificity, PAA-CeO2 was modified with protein-G for further attachment of antibodies. The authors conjugated this platform with antifolatereceptor antibody, which can recognize the overexpressed folate receptors in many tumors. Lung cancer cell line A-549 was used as a model target, and the fluorescent intensity of resorufin was correlated with the number of cancer cells (Figure 2F). Breast carcinoma cells (MCF-7) were used as a control (Figure 2G), which do not overexpress the folate receptor. Typical immunoassays require conjugation of an enzyme or nanozyme with the recognition antibody, and tedious washing steps to decrease nonspecific binding. Coating of a thick surface antibody layer may also inhibit the catalytic activity of nanozymes. A
more convenient method is by coupling nanozymes with natural enzymes, and the conjugate may have high activity and selectivity towards a wide range of targets. In a typical design, the product of a natural enzyme can serve as the substrate of the linked nanozyme. In this regard, H2O2 is a common intermediate, serving as the product of several natural oxidases, for instance, glucose oxidase (GOx) , cholesterol oxidase , xanthine oxidase , and urate oxidase . Wei et al reported this strategy using GOx and Fe3O4 NPs to detect glucose with a LOD of 30 µM and a linear range of 50 µM to 1 mM. To avoid side reactions of intermediates, nanozymes and protein enzymes need a more precise control over their spatial positions. Integrated nanozymes was such a platform to facilitate the diffusion of intermediates . 3.2 MONMs for target recognition Aside for signaling, MONPs can also recognize certain analytes, for example, by adsorbing or reacting with ligands. A representative example is that MnO2 nanosheets as an oxidant can be reduced into Mn2+ by reducing agents. In 2011, Deng et al  combined MnO2 with lanthanide-doped upconversion nanoparticles (UCNPs) for intracellular GSH detection. Compared to organic fluorophores, UCNPs have sharper emission spectra, which could be useful for multiplexed detection. To deposit UCNPs on MnO2 nanosheets, KMnO4 was added into core-shell NaYF4:Yb/[email protected]
nanoparticles using 2-ethanesulfonic acid (MES) as a reducing agent. Since the absorption spectra of MnO2 overlaps with the emission of UCNPs, the authors believed FRET gave rise to the quenching. MnO2 can react with glutathione (GSH) and convert into Mn2+ ions. With increasing GSH concentration, the luminescence of UCNPs was gradually recovered up to 100-fold, which was consistent with the dissolution of MnO2.
This assay was also very selective, and several amino acids, glucose, fructose, BSA did not produce a signal. Furthermore, the nanoconjugates were delivered into cancer cells to achieve intracellular GSH detection. Similar to this work, fluorescent nanomaterials, such as graphitic-phase C3N4 , carbon dots , boron nitride quantum dots , and gold nanoclusters  and small molecule fluorophores  have also been used to sense GSH with MnO2. However, this assay may not be sufficiently selective since MnO2 could also react with other reducing agents or ligands, such as ascorbic acid [59,60], cysteine , and H2O2 . Aside from dissolution of MONMs, target analytes may simply displace pre-adsorbed surface ligand and generate an optical signal. Such a strategy has been applied to sense various anions, H2O2, and small molecules, which will be discussed later. 3.3 MONMs for both recognition and signaling Intrinsic optical properties. MONPs possess special optical properties, which are sensitive to surface chemistry. For example, nanoceria dispersion is almost colorless, but it immediately turns yellow then dark orange with H2O2 (Figure 3A,B) . This color change was ascribed to the increased percentage of surface Ce(IV), and surface peroxide species. This colorimetric assay showed a linear range from 0.01 to 0.15 mM for detecting H2O2. Furthermore, a paper-based assay was designed by attaching nanoceria on silanized filter paper with aminopropyltriethoxysilane (APTS) (Figure 3C). This assay showed a linear range from 2.5 mM to 100 mM H2O2. When combined with glucose oxidase, glucose can also be detected. The color change of nanoceria however is not unique to H2O2. Several antioxidants, including ascorbic acid (AA), gallic acid (GA), vanillic acid (VA),
epigallocatechin gallate (EGCG), quercetin (Q), and caffeic acid (CA) have been shown to coordinate with nanoceria and also resulted in a strong color change  (Figure 3D). For example, dopamine also bind to nanoceria and gave a dark color . Therefore, application of this method can only be in specialized cases where the sample matrix contains only one type of these molecules. Nanozymes can be integrated with luminescent properties to light up the analyte binding. Pratsinis et al reported an Eu3+-doped nanoceria (CeO2:Eu3+) possesses both catalase-like activity and luminescence property with an emission peak at ca. 590 nm. The rapid adsorption of H2O2 by nanoceria resulted in the decrease of CeO2:Eu3+ luminescence. With this turn-off sensor, highly sensitive detection of H2O2 was demonstrated both in buffer (LOD 150 nM) , and in a bacterial cell culture .
Figure 3. MONMs as both recognition and signaling elements. (A) A schemes and photos of nanoceria (B) in buffer and (C) deposited on filter paper showing the color change by H2O2. (D) Color change of nanoceria dispersion with addition of antioxidants, ascorbic acid (AA), gallic acid (GA), vanillic acid (VA), epigallocatechin gallate (EGCG), quercetin (Q), and caffeic acid (CA). (E) A scheme showing that analytes
interfacing with nanozymes may alter its enzymatic properties. (F) A scheme of fluoride boosted the oxidase-like activity of nanoceria. (G) Photos showing the oxidation of ABTS with nanoceria and various concentrations of fluoride. (H) Selectivity test of different anions on oxidase activity of nanoceria using ABTS as a substrate. Panel (B-C) reprinted from ref.  with permission, copyright 2011 American Chemical Society, (D) from ref.  with permission, copyright 2012 Royal Society of Chemistry, (F-H) reproduced from ref.  with permission, copyright 2016 Royal Society of Chemistry.
Tuning the catalytic activity of metal oxides. Tuning the surface chemistry of MONMs can dramatically change their nanozyme activity, which can also be used to detect the promotor or inhibitor (Figure 3E). For example, we reported that fluoride can accelerate the oxidase-mimicking activity of nanoceria up to 100-fold (Figure 3F) . The catalytic capability was evaluated using the steady-state kinetics of TMB and ABTS oxidation. We found the catalytic efficiency (kcat/Km) of nanoceria was enhanced by 15fold and 100-fold for ABTS and TMB, respectively. Mechanistic studies showed that fluoride capping altered the surface charge, and oxygen vacancy levels of nanoceria. With this enhancement effect, we were able to detect fluoride concentration down to 10 µM by naked eyes (Figure 3G) and 0.64 µM by a spectrometer. This assay was highly selective for fluoride, while common anions such as chloride did not produce a signal (Figure 3H). Finally, we showed fluoride in toothpastes can be accurately quantified. The oxidase-like activity of nanoceria can be also be modulated by nucleoside triphosphates (NTPs). Xu et al  reported NTPs functioned as a cofactor, and its hydrolysis may further promote the oxidative reactions catalyzed by nanoceria. Among
the four NTPs, GTP was most efficient, then ATP, UTP, and CTP. Based on the differential enhancing effect, the authors designed a colorimetric assay to type singlenucleotide polymorphism. Many of the abovementioned discoveries are difficult to predict and some were from random discoveries in the lab. Different results may come up in the same nanozyme system, which could be attributable to different experiment conditions such as buffer, pH and concentration. Therefore, careful studies and systematic tests are needed to fully understand these systems. At the same time, such studies can offer interesting insights into the surface and catalytic properties of MONMs.
4. Sensors based on DNA-functionalized MONMs DNA oligonucleotides with defined lengths and sequences can be chemically synthesized with high stability. During this process, many modifications can be added for bioconjugation and signaling. Recent developments in using DNA as a material has also inspired its research on biointerfaces. In addition, various DNA sequences with molecular recognition (aptamers) and catalytic (DNAzymes) functions have been isolated. In this section, we review the current understandings on DNA-MONPs interactions and outline several strategies using DNA-MONPs to design assays to probe specific targets and surface reactions. 4.1 Adsorption of DNA on MONMs Since appropriate anchoring of DNA on MONMs is often the first step in designing DNA-MONM sensors, it is important to understand the adsorption behavior of DNA. Adsorption of nucleic acids on metal oxides has been researched over years, and early
studies were concerned with double-stranded nucleic acids (over 1000 bp) on mineral surface to investigate the prebiotic chemistry . For sensing purpose, single-stranded (ss)DNA less than 100 bases are more frequently used. Study of adsorption of DNA oligonucleotides by a large number of MONMs have been performed, including TiO2 , In2O3 , ZnO , CeO2 , Fe3O4  NiO , CoO(OH) [76,77], MnO2 [78,79], HfO2 , Al2O3 [81,82], and Mg/Fe double hydroxide . Adsorption of DNA on MONMs takes place mostly on the phosphate backbone via coordination with non-saturated surface metals and/or electronic interactions. Using ligand displacement assays, we confirmed that phosphate-binding predominates in oxides tested . With Raman spectroscopy, Tian et al proved that all four DNA bases bind to the Al2O3 via the phosphate backbone . However, contributions from nucleobase adsorption cannot be fully excluded. Using photoelectron and X-ray absorption spectroscopies, adenine bonding with CeO2 was observed . With fluorophore-labeled DNA, we studied the adsorption kinetics. The kinetics mainly depends on the surface charge of MONMs. For examples, at physiological pH, several MONMs are negatively charged (Table 1), thus repelling DNA strands. Increasing the ionic strength could screen the long-ranged charge repulsion. Once adsorbed, DNA on MONMs are quite stable against changes in salt concentration , and interferences from proteins . However, anions and ligands having high affinity with MONMs can effectively release the DNA, which is the basis of several displacement-based assays (vide infra). One problem of MONMs is difficulties of covalent modification. Most of the works relied on physiosorbed biomolecular probes. While physisorption is simple and
cost-effective, it is more difficult to achieve consistent measurements especially in a complex environment. Therefore, one of the directions is to achieve stable ligand conjugation. We recently developed an indirect method by preparing DNA-functionalized gold nanoparticles called spherical nucleic acids (SNAs) . Then, the SNA can extremely tightly adsorb on metal oxide (and other types of inorganic nanomaterials) via polyvalent interactions. 4.2 Assays based on competitive adsorption As discussed in section 3, MONPs can selectively adsorb a lot of molecules, which could be used as potential sensors. However, the adsorption process typically does not generate optical signals. DNA oligonucleotides modified with fluorophores have been widely used in biosensing [86,87]. By controlling the length and sequence of DNA, this system can systematically tune the interaction strength and performance of related sensors. Detecting anions. Magnetic iron oxide nanoparticles have been used to adsorb aqueous arsenic species for decades. However, such a process is difficult to probe due to the lack of a convenient signaling mechanism. Combining the fluorescent quenching and DNA adsorption abilities of Fe3O4 NPs, we designed a fluorescent assay to quantify As(V) in water . The assay was based on the competitive adsorption of As(V) with 6carboxyfluorescein (FAM)-modified DNA on the Fe3O4 surface (Figure 4A,B). This fluorescence turn-on sensor was able to detect As(V) down to 300 nM (130 nM if As(V) was pre-adsorbed to inhibit DNA adsorption). By optimizing the DNA length and sequence, and oxide surface, Lopez et al demonstrated that the limit of detection can be further lower by 10-fold using FAM-C5 DNA on CeO2 NPs . This displacement assay was selective over a few major anions, expect for phosphate, which is very similar to
arsenate. The displacement assay has been further used to probe the adsorption of selenite and selenate , phosphite species with different oxidation states , and phosphate species with different lengths [91,92]. A key problem is selectivity (e.g. hard to discriminate phosphate and arsenate). We proposed that a sensor array may help discriminate the very similar anions, such as phosphate, arsenate, and arsenite . The simple sensing mechanism was applied on 19 different MONMs to screen for optimal sensing candidates. Finally, CeO2, Fe3O4, and ZnO were used to form a sensor array to distinguish the three anions using the principle component analysis (PCA). Shorter DNA adsorbs more weakly on surfaces, but single nucleotides can also be tightly adsorbed in some cases when the nucleotide contains a triphosphate (e.g. ATP). Tseng and co-workers  used a fluorescent small molecule, boron dipyrrometheneconjugated adenosine 5'-triphosphate (BODIPY-ATP) as a probe to detect ATP and pyrophosphate (Figure 4C,D). The fluorescent BODIPY analogue shows a high extinction coefficient (>80 000 cm−1 M−1) and high quantum yield in water. The triphosphate part of ATP can coordinate with the Fe3+/Fe2+ on Fe3O4 NPs surface. The authors proposed that the Fe3+ sites played a major role in the fluorescence quenching via the photoelectron transfer pathway. The competitive adsorption of ATP or pyrophosphate on Fe3O4 NPs released the BODIPY-ATP (Figure 4C). With this strategy, the authors were able to detect ATP and pyrophosphate down to 7 and 30 nM, respectively. A diluted blood sample was also tested. In all three spiked samples, high recovery efficiencies were achieved (90%-109%). The Tang group also used BODIPY-ATP and nanoceria to detect arsenate . The assay was very sensitive (LOD 7.8 nM) and selective, even phosphate
did not cause interference. Detection of arsenate in real water samples were also demonstrated.
Figure 4. Sensors based on the competitive adsorption with DNA on MONMs. Schemes and fluorescent images showing the detection of (A) arsenate on Fe3O4, (C) ATP on Fe3O4, and (E-F) H2O2 on CeO2. The fluorescence response as a function of analyte concentration for (B) arsenate, (D) ATP, and (G) H2O2. The insets show the initial linear responses. Panel (A,B) reprinted from ref.  with permission, copyright 2014 Royal Society of Chemistry, (C,D) from ref.  with permission, copyright 2013 American Chemical Society, and (E-G) from ref  with permission, copyright 2015 American Chemical Society.
Detection of H2O2. Nanoceria as catalase-mimic can bind H2O2 rapidly and change color to yellow-orange. However, this process is not very sensitive (LOD in the low mM range). We used fluorescently labeled DNA oligonucleotides to probe this fast and high affinity binding (Figure 4E,F,G) . Upon adding H2O2, the quenched fluorescence of DNA was immediately recovered (Figure 4F). Compared with the 22
colorimetric assay, our fluorescence assay was much more sensitive (LOD = 130 nM). More importantly, we revealed that the signaling mechanism was based on simple displacement. This assay was also very selective towards common metabolites, including some amino acids, sugars, and neurotransmitters. The assay can be further combined with GOx to achieve the detection of glucose level in serum samples. The Tang group has also used this method to detect H2O2 in cells, and woundinduced oxidative damages , and to deliver RNA into cells . This assay is not limited to fluorescent probes. Qi et al  reported a liquid-crystal (LC)-based platforms for H2O2 and glucose. Non-labeled DNA was displaced by H2O2 from nanoceria, and it would interact with the positive layer (cationic surfactants) on a LC surface. This DNAsurfactant interaction disrupted the orientation of the LCs, resulting in an optical signal from dark to bright. The assay showed high sensitivity (LOD 28.9 nM H2O2) and selectivity.
4.3 DNA-modulated nanozyme activity DNA adsorption may alter the nanozyme-like activity of MONMs in different ways (Figure 5A). Nanoceria as an oxidase-mimic requires direct interaction with its substrate for the catalysis. We found that the oxidase activity of nanoceria was inhibited after attaching DNA on its surface based on the TMB oxidation (Figure 5B) . Control experiments showed the decrease of activity correlated with the DNA adsorption process (Figure 5C). Compared to other synthetic polymers, DNA inhibited the oxidase activity quite efficiently. Anionic PAA and neutral polyethylene glycol (PEG) did not show inhibition effect. Furthermore, it required up to 2% of polystyrene sulfonate (PSS) to
completely inhibit the activity, while only 0.004% of DNA could achieve the same effect. The strong binding between the DNA backbone and nanoceria may block the active site for substrate approaching, and thus slow down the catalytic process. Different from our observation, Andreescu and co-workers found that non-labeled DNA aptamer for ochratoxin A (OTA) adsorbed by nanoceria can stabilize the dispersion and increase the oxidase-like activity for oxidation of TMB . If the aptamer formed a complex with its target, the adsorption of the aptamer to CeO2 might be impeded and the bare nanoceria aggregated with a lower activity. Using the OTA aptamer, the authors were able to detect OTA with a LOD of 0.15 nM, and a dynamic range from 0.2 to 3.75 nM. This assay was quite selective to OTA since OTB and other analytes did not show any difference in TMB oxidation. Also, OTA did not show any effect using a random DNA sequence on nanoceria. We noted that the authors used phosphate buffer and phosphate can strongly adsorb on nanoceria. The buffer difference could explain the different effects of DNA. Such controversy on the effect of DNA has also been observed on a peroxidase mimic nanozyme, Fe3O4 NPs. Park et al  reported that the peroxidase-like activity of Fe3O4 NPs was inhibited by DNA from polymerase chain reaction amplification. Using o-phenylenediamine (OPD) as a substrate, the authors revealed that the negatively charged DNA may bind non-specifically to the positive substrate, and then block its surface to inhibit oxidation. However, we observed that the Fe3O4-catalyzed TMB oxidation by H2O2 could be enhanced in the presence of DNA oligonucleotides (Figure 5D), and the enhancement effect was positively correlated with DNA concentration (Figure 5E) . Similar to the previous finding, DNA as a negative polymer may bind
to TMB. We reason that DNA oligonucleotides on Fe3O4 cannot fully block the access of small H2O2. At the same time, adsorbed DNA turned the surface to negatively charged for efficient adsorption of positively charged TMB substrate. Indeed, oxidation of negatively charged ABTS was inhibited due to electrostatic repulsion. Recently, Zeng et al performed a systematic study on various DNA secondary structures, including short dsDNA, ssDNA, hairpin DNA and hybridization chain reaction (HCR) products (similar to long dsDNA) . They found that the largest rate enhancement was with the HCR products. Based on such enhancement effect, the authors developed a colorimetric assay for DNA detection with high sensitivity (LOD 0.1 nM).
Figure 5. (A) Regulating the nanozyme-activity of MONMs by DNA. (B) Photographs showing the oxidation of TMB by nanoceria without and with DNA. (C) Decrease of absorbance at 652 nm indicating decreased oxidation activity (y-axis on the left) and 25
fluorescence from non-adsorbed DNA (y-axis on the right) as a function of DNA concentration added into nanoceria. (D) Photographs showing oxidation of TMB by H2O2 catalyzed by Fe3O4 NPs without and with DNA. (E) Kinetics of TMB oxidation by Fe3O4 and H2O2 by monitoring the absorbance at 652 nm as a function of DNA concentration. Panel (B,C) reprinted from ref.  with permission, copyright 2013 American Chemical Society, and (D,E) from ref.  with permission, copyright 2016 Royal Society of Chemistry.
4.4 Assays based on DNA recognizing analytes DNA oligonucleotides can selectively bind a wide range of analytes, including complementary nucleic acids, metal ions, small molecules, proteins, and even cells. [86,87,103] Combining these functional DNAs with MONMs greatly expands the sensing functions. A typical design requires two features of MONPS. First, it should quench fluorophores on or near the surface. We tested the DNA adsorption and fluorescence quenching properties of 19 different MONMs , and found 10 materials were able to adsorb ssDNA and function as efficient quenchers, including CeO2, CoO, Cr2O3, Fe2O3, Fe3O4, Mn2O3, NiO, and TiO2 (anatase). Second, the surface should be able to discriminate probe DNAs (non-structured) and probe-target complexes (e.g. duplex, aptamer-analyte complex) (Figure 6A). While most MONMs adsorb DNA through the phosphate backbone, they may still have differential ability in adsorbing ssDNA and dsDNA, considering the difference in flexibility between ssDNA and dsDNA. For example , TiO2 can even differentiate 1 nM cDNA (Figure 6B) .
He et al reported MnO2 nanosheets can effectively adsorb ssDNA but not dsDNA . The fluorescence quenching ability of MnO2 made it possible to design DNA-based biosensor. With increased cDNA concentration, the fluorescence quenched by MnO2 reported that was lower, and a linear range from 0 to 5 nM was obtained with a LOD of 0.3 nM. Similarly, adenosine with a LOD of 5 µM was achieved if an aptamer was used. Liu and co-workers developed a fluorescence turn-on biosensor for thrombin by attaching the thrombin aptamer on bare Fe3O4 NPs . The sensor can detect thrombin with high sensitivity (LOD 0.5 nM). More importantly, the authors showed this assay can detect thrombin in the presence of interference proteins, and the recovery efficiency was between 91% and 104%. Song and co-workers developed a platform for detecting multiple targets using the pristine α-Fe2O3 NPs . DNA probes labeled with various organic dyes (FAM, cyanine dye 3 (Cy3), and 6-carboxy-X-rhodamine can be immobilized and all three fluorophores can be quenched. They further demonstrated that three targets (ssDNA, miRNA, and dsDNA) can be detected with high sensitivity, simultaneously (Figure 6C-6E). Similar sensing strategies have been demonstrated on nanoceria for DNA detection , on MoO3 nanosheets for prostate specific antigen , and on CoOOH sheets for T4 polynucleotide kinase activity . Non-convalent DNA sensing was first reported on the graphene oxide (GO) surface , and the concept has been expanded to other surface, including MONMs. One critical issue is the nonspecific displacement of DNA by multiple specie. While DNA-GO and DNA-MONMs showed similar sensing performances in cDNA induced signal recovery, DNA binds to GO and MONMs through bases and phosphate, respectively (Figure 6F). Such orthogonal adsorption dramatically changed the stability
of DNA on nanomaterials in biological complex environment. For examples, BSA, an abundant serum protein caused significant false-positive signals by competing with DNA bases on GO (Figure 6G) . However, DNA adsorbed on MONMs (e.g. NiO in Figure 6H) showed much less non-specific displacement. As a result, the signal-to-background ratio of DNA-NiO was 12-fold higher than that of DNA-GO, showing promising values in practical applications.
Figure 6. Sensing based on DNA recognition. (A) A scheme showing the MONMs can adsorb ssDNA but not dsDNA or aptamer-analyte complexes. (B) Kinetic traces of DNA adsorption by TiO2 NPs in the presence of different concentrations of cDNA. (C-E) Linear ranges of fluorescence response of DNA-Fe2O3 NPs in detecting (C) ssDNA, (D) 28
miRNA, and (E) dsDNA. (F) A scheme showing the orthogonal adsorption of DNA on GO by bases and on MONMs by phosphate. Kinetics of DNA recovery by adding cDNA in the presence of excess BSA when probe DNA was adsorbed on (G) GO, and (H) NiO NPs. Panel (B) reprinted from ref.  with permission, copyright 2012 American Chemical Society, (C-E) from ref.  with permission, copyright 2015 Elsevier B.V., and (F-H) from ref.  with permission, copyright 2018 Royal Society of Chemistry.
5. Conclusions and future perspectives In this review, we summarized the strategies of using MONMs for developing sensors and biosensors. In addition, related fundamental surface chemistry of MONMs and their adsorption of DNA are also described. Some MONMs alone can be sensors since they have catalytic activities that can be modulated by target analytes. Many oxides can achieve competitive adsorption between a signaling molecule/particle and target molecules to produce optical signals. In addition, DNA aptamers and other DNA probes can also be functionalized on MONMs to detect their targets. The strategies for MONMs can in principle be used for other types of materials such as gold and carbon. However, metal oxides also have their own properties, complementary to gold and carbon. For example, DNA adsorbs on gold and carbon based surfaces mainly via its nucleobases, while DNA uses its phosphate backbone on MONMs. A unique advantage is that there are many choices of the metal species, allowing distinct magnetic, catalytic and optical properties for different applications. We believe metal oxides represent a unique class of material for sensor development and its value just start to be appreciated. However, a
number of problems need to be addressed before this system can be of practical useful. These problems have defined some future research opportunities for this field. First, for competitive assays, the biggest challenge is specificity. Since recognition often relied on the surface of metal oxide instead of more specific biomolecules, any competitive molecules can produce a signal. To overcome this problem, we need to find appropriate application scenario where the potential interfering species are minimal. In addition, sensor arrays can be prepared to maximizing the selectivity. To be most efficient, the number of metal oxides used should be kept to a minimal, and this would require close to orthogonal response of different analytes to different oxides. Second, metal oxides can have different crystalline forms and sizes. These properties are likely to affect the activity of the oxides in terms of both catalysis and adsorption. The most studies material on this front is probably nanoceria, where smaller nanoceria is often more catalytically active than larger ones. Systematic investigation on other metal oxide materials will likely produce another level of control and better sensors. Third, bioconjugation is key. For optimal biosensors, it is important to have active biomolecules linked to metal oxide surfaces. Unlike other surfaces such as gold and graphene oxide, linking biomolecules directly to metal oxides is quite challenging, especially when we need to maximally retaining surface accessibility. In most cases, conjugation was achieved by first coating the oxide with a layer of polymer and the intended biomolecules are then linked to the polymer layer. Developing new chemistries for directly grafting DNA and antibodies on metal oxide is another major challenge and opportunity to make breakthroughs.
Fourth, metal oxides have been used as dispersed nanoparticles in most of these cases. As far as their catalytic activities are concerned, smaller particles are more active due to the presence of more surface defects and larger specific surface areas. Making them into bulk planar surfaces may decrease their catalytic activities. Since catalytic activity is a major component of metal oxide based sensors, this is different from goldbased sensors, which mainly rely on their optical properties. Therefore, another challenge is to make such assays into stable kits and interface them with measurement devices. Most of the analytical work has been focused on detection in clean buffers or spiked samples. Their applications in real samples have to be the real test. A lot of the sensing is from the metal oxide surface adsorption of target molecules or ions, and the specificity is not very high sometimes. Rational combination of metal oxides with biological ligands such as aptamers and even molecularly imprinted polymers could be a useful direction. Finally, most studies described here used only the redox catalytic activities of metal oxides. It is highly desirable to expand to other types of reactions for more versatile applications.
Acknowledgements Funding for the related work in the Liu lab was mainly from the Natural Sciences and Engineering Research Council of Canada (NSERC).
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Metal oxide nanomaterials serve for both target recognition and signaling purposes.
Surface modification affects the catalytic properties of metal oxide nanoparticles.
DNA functions as fluorescent probe for the surface reactions on metal oxides.
DNA-metal oxide conjugates as tunable hybrid materials for biosensing.