Nanomaterial-Supported Enzymes for Water Purification and

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Nanomaterial-Supported Enzymes for Water Purification and Monitoring in Point-of-Use Water Supply Systems Published as part of the Accounts of Chemical Research special issue “Water for Two Worlds: Urban and Rural Communities”. Meng Wang, Sanjay K. Mohanty, and Shaily Mahendra*

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Department of Civil and Environmental Engineering, University of California, Los Angeles, California 90095, United States CONSPECTUS: Increasing pollution of global water sources and challenges in rapid detection and treatment of the wide range of contaminants pose considerable burdens on public health. The issue is particularly critical in rural areas, where building of centralized water treatment systems and pipe infrastructure to connect dispersed populations is not always practical. Point-of-use (POU) water supply systems provide cost-effective and energy-efficient approaches to store, treat, and monitor the quality of water. Currently available POU systems have limited success in dealing with the portfolio of emerging contaminants, particularly those present at trace concentrations. A site-to-site variation in contaminant species and concentrations also requires versatile POU systems to detect and treat contaminants and provide on-demand clean water. Among different technologies for developing rapid and sensitive water purification processes and sensors, enzymes offer one of the potential solutions because of their strong activity and selectivity toward chemical substrates. Many enzyme−nanomaterial composites have recently been developed that enhance enzymes’ stability and activity and expand their functionality, thus facilitating the application of nanosupported enzymes in advanced POU systems. In this Account, we highlight the strengths and limitations of nanosupported enzymes for their potential applications in POU systems for water treatment as well as detection of contaminants, even at trace levels. We first summarize the mechanisms by which silica, carbon, and metallic nanosupports improve enzyme stability, selectivity, and catalysis. The unique immobilization properties and potential advantages of novel bioderived nanosupports over non-bioderived nanomaterials are emphasized. We illustrate prospective applications of nanosupported enzymes in POU systems with different roles: water purification, disinfection, and contaminant sensing. For each type of application, nanosupported enzymes offer higher performance than free enzymes. Nanosupports prolong enzymes’ lifetimes and improve the rates of contaminant removal by concentrating contaminants near the enzymes. Nanosupports also stabilize antimicrobial enzymes while facilitating their attachment to bacterial surfaces, thereby increasing their potential uses for disinfection and prevention of biofouling in water purification and storage devices. As enzyme-based electrochemical sensors rely on electrochemical reactions of enzymatically generated species, the ability of conductive nanosupports to enhance enzyme activity and stability and to promote transfer of electrons onto the electrode greatly improves the sensitivity and durability of electroenzymatic contaminant sensors. Despite the promising results in laboratory settings, the application of nanosupported enzymes in real-world POU systems requires the implementation of multiple enzyme combinations and strategies for minimizing health risks associated with unintended releases of nanomaterials. Finally, we identify multidisciplinary research gaps in the development of nanosupported enzyme treatment systems and provide frameworks for the early adopters to make informed decisions on whether and how to use such POU systems.

1. INTRODUCTION

footprints to provide millions of rural communities with access to safe drinking water.2,3 However, the increasing occurrence of emerging contaminants (e.g., endocrine disruptors, pesticides, and pharmaceuticals), heavy metals, and pathogens are challenging current treatment technologies, including POU systems.4 Meanwhile, POU systems typically use reactive materials to adsorb or degrade a specific set of contaminants, and the efficiency of POU systems relies on prior knowledge of types of contaminants to be treated and their ability to remove

Water, an essential ingredient for life, shapes human history and civilization. Despite progress in water purification technologies in the last century, 2.1 billion people still lack access to safely managed drinking water, and by 2025, half of the world’s population will live in water-stressed areas.1 Connecting every location by pipe networks or other water infrastructure is expensive and not practical, particularly in rural and remote areas, where populations are geographically dispersed and migrating. In contrast, point-of-use (POU) water treatment systems can provide sustainable, resilient, less expensive, and flexible methods with low environmental © XXXX American Chemical Society

Received: November 30, 2018

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Figure 1. Summary of various nanosupports used in enzyme immobilization. (A) TEM image of chemically synthesized mesoporous silica with a pore size around 10 nm. (B) SEM image of silica synthesized from a lysozyme-directed biosilicification process. (C) Various carbon nanostructures including fullerene, CNT, and graphene, and a schematic of nanogel synthesis. (D) Illustration and TEM image of an enzyme-embedded zeolitic imidazolate framework. (E) SEM images of protein−copper hybrid nanoflowers. (F) Illustration and TEM image of vault nanoparticles (PDB entry 6BP8). Seventy-eight copies of the major vault protein are colored in blue and gold alternatively, with one single chain colored red. (G) Schematic of protein encapsulation into bacteriophage P22 capsid. (H, I) Illustration and AFM image of a DNA origami box encapsulated with proteins. Reproduced with permission from (A) ref 12, copyright 1992 American Chemical Society; (B) ref 44, copyright 2006 Wiley; (D) ref 16, copyright 2014 American Chemical Society; (E) ref 17, copyright 2012 Nature Publishing Group; (F) ref 24, copyright 2018 Wiley; (G) ref 29, copyright 2014 Royal Society of Chemistry; (I) ref 34, copyright 2012 American Chemical Society.

enzyme-based technologies in water supply systems are lack of stability and degradation of enzymes under conditions expected in drinking water sources.9 Recent advances in nanotechnology have led to highly active and robust enzyme− nanomaterial composites, many of which exhibit better performance than free enzymes.10 Nanosupports improve stability and activity of immobilized enzymes, expand their applicability, and provide manipulation platforms that facilitate transfer of the methodology to field applications. Many studies to date have explored nanosupported enzymes toward bioremediation, medical applications such as therapeutic delivery and disease diagnosis, and industrial production of biorenewables, cosmetics, and food additives,4,6,10 whereas much less attention has been given to the development of nanosupported enzyme-enhanced POU water supply systems. The aim of this Account is to highlight the strengths and limitations of nanosupported enzymes in water purification and monitoring so that it can inform the technological advances in the development of nanosupported enzyme systems and provide a framework for adoption of the technologies in existing POU systems. We summarize recent developments of enzyme nanosupports, focusing on silica, carbon, metals, and bioderived materials (Figure 1), and discuss how they improve

the wide range of contaminants simultaneously. The fluctuations of contaminant species and levels in source waters and the lack of diagnosis of local contamination and treatment goals present barriers to customization of POU systems to provide targeted treatment.5 Hence, there is a need to develop portable technologies to detect a broad range of contaminants in water and tailor the POU systems to remove those pollutants. Enzymes are biological catalysts that mediate biochemical reactions with high efficiency. As a result of their extensive exploration in bioremediation in the past few decades, many enzymes such as laccases, carboxylesterases, haloalkane dehalogenases, and lysozymes have been identified for the removal of contaminants and pathogens.4,6,7 Enzyme-based water treatment is rapid, sensitive, and sustainable, and it has shown improved performance over contemporary technologies in many studies.4 It is also a versatile process, in which enzymes can be customized to meet treatment demands at various locations. Moreover, the development of high-performance sensors based on the specific interactions between enzymes and contaminants can enable rapid on-site diagnosis of contaminants and inform the design of POU systems for targeted treatment.8 However, challenges for implementing B

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Accounts of Chemical Research 2.3. Metallic Nanosupports

enzyme performance. The mechanisms by which nanosupported enzymes can advance removal and monitoring of chemical pollutants and pathogens are also described. Finally, potential gaps and limitations are identified, and recommendations are provided for future studies in order to facilitate the effective use of nanosupported enzymes in POU systems with a broader aim to minimize water supply issues in rural and remote areas.

Metal−organic frameworks (MOFs) are ordered porous metallic nanomaterials that are synthesized by cross-linking of metal ions and organic ligands.15 Immobilization of enzymes onto MOFs can be achieved by physical adsorption or covalent binding between enzyme molecules and built-in functional groups on MOFs.15 Recently, a one-pot synthesis process in which MOFs are self-assembled around enzyme molecules was reported.16 Protein−inorganic hybrid nanoflowers represent another class of metallic nanosupports. The first was synthesized via a single-step coprecipitation of copper salts and proteins,17 and this was subsequently extended to other metal ions such as iron.18 One benefit of MOFs and hybrid nanoflowers is that they can create a microenvironment with elevated concentrations of metal ions, which might synergistically interact with immobilized enzymes to boost their activities, particularly for metal-containing enzymes.16−18 For easy separation and recovery of enzymes from the reaction media, magnetic nanoparticles have been used to immobilize enzymes. 10 Attempts have been made to immobilize enzymes on noble-metal-containing nanoparticles too. Biological synthesis, dendrimer-assisted synthesis, and many other methods have been developed to control the size and morphology of noble metal nanomaterials.19 Although noble metals are less likely to be used in water treatment practices because of high cost, their aggregation-state-dependent optical properties coupled with enzyme activities are useful for fabricating sensors for the detection of trace contaminants.20

2. NANOSUPPORTS FOR ENZYME IMMOBILIZATION 2.1. Silica Nanosupports

Silica (SiO2), a chemically and thermally inert, abundant material, is the most widely used among solid supports developed for enzyme immobilization. Mesoporous silica materials synthesized from silicon alkoxide precursors via surfactant-templated polymerization processes are of particular interest, as they possess large surface areas (300−1200 m2/g) and pores that can be tailored from 1.6 to 30 nm to anchor proteins of different sizes.9,11,12 The bare mesoporous silica can directly adsorb enzymes or be chemically grafted to introduce functional groups such as amines, thiols, and silanols to covalently bind the enzymes. On the other hand, in situ synthesis of silica in the presence of enzymes can also entrap enzymes. Surfactant-templated silica synthesis is often conducted under harsh conditions such as extreme pH, high temperature, and organic solvents, which can result in significant enzyme activity loss. The use of biomolecules to induce silica precipitation, known as biosilicification, at neutral pH in water solutions provides an alternative enzyme entrapment approach.13 Cationic biomolecules, including natural proteins that have basic isoelectric points, synthetic polycationic peptides, and block copolypeptides, have been identified as effective precipitants.13

2.4. Bioderived Materials as Nanosupports

Production of synthetic nanosupports typically involves toxic substances and organic solvents, and some of these supports, such as CNTs and graphene, can be cytotoxic. In contrast, bioderived materials are generally more environmentally friendly and biocompatible. They can be synthesized in vitro or in vivo through biological activities under physiological conditions, generating minimal hazardous wastes. 2.4.1. Protein Cages. Nature provides various types of supramolecular protein structures, including fibers, rings, tubes, catenanes, knots, and cages.21 Among them, protein cages have gained particular attention for enzyme immobilization, as they not only have large core volumes to hold enzyme molecules but also protect and stabilize immobilized enzymes in protein shells. Vaults are the largest cytoplasmic ribonucleoprotein particles in nature and are assembled from 78 copies of major vault protein along with other proteins and RNA fragments.22 Heterologous expression of major vault protein leads to the formation of recombinant vaults that have large cores to anchor multiple enzyme molecules per particle.23,24 By genetic attachment of a vault-interacting domain (named INT) to the enzyme, the protein complex can be directed and anchored on the inner surface of vaults.25 As this process depends on the specific interactions between the INT and vaults, vaults selectively take up INT-fused enzymes from crude enzyme extracts, which saves the cost of enzyme purification. Lumazine synthase,26 ferritin,27 and encapsulins28 are three other examples of natural protein microcompartments that utilize specific peptide tags to direct and sequester enzymes in the cage cores. Virus-like particles are viruses that are devoid of their nucleic acids and contain only noninfectious protein capsids such as

2.2. Carbon Nanosupports

Carbon nanosupports such as carbon nanotubes (CNTs) and graphene immobilize enzymes through physical adsorption and surface grafting, where enzymes are covalently bound to carbon nanosupports.10 Mesoporous carbon materials are also used as supports for enzyme immobilization.9 They are synthesized via polymerization and carbonization of carbon precursors on templates such as mesoporous silica and block copolymers, followed by template removal. Compared with silica supports, carbon materials have extraordinary electrical conductivity and can promote electron transfer reactions on electrodes, thus providing great advantages in developing enzyme-based amperometric sensors.9,10 Another method to prepare carbon nanosupports is the nanogel approach, which constructs a thin and porous polymer layer surrounding enzyme molecules through a two-step procedure.14 In the first step, ethenyl groups are introduced on the enzyme surface by modifying amine groups in amino acid residuals. Subsequently, polymerization at the ethenyl groups occurs after addition of monomers, cross-linkers, and initiators, resulting in polymer-trapped single enzyme molecules. Nanogel encapsulation minimally affects the activity of the enzymes while strongly preserving their robustness even under harsh conditions. Hence, it enables subsequent manipulation of enzymes such as further immobilization under extreme conditions that are fatal to free enzymes.14 C

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Accounts of Chemical Research Table 1. Summary of Nanosupports and Their Advantages and Potential Applications in POU Systems nanomaterial

advantages

potential applications

refs

Silica Nanosupports chemically synthesized silica

easy synthesis

biosynthesized silica

milder synthesis conditions less enzyme activity loss

CNTs, graphene, and mesoporous carbon nanogels MOFs and protein−inorganic nanoflowers magnetic nanoparticles noble metal nanoparticles

protein cages

DNA origami

Carbon Nanosupports extraordinary electric conductivity

minimal enzyme activity loss Metallic Nanosupports increased enzyme activity

contaminant removal disinfection contaminant removal disinfection

13, 38, 44

enzymatic amperometric sensors disinfection contaminant removal

40, 43, 46, 50

contaminant removal

16−18

easy recovery contaminant removal unique aggregation-state-dependent optical property enzymatic colorimetric sensors enhanced quantum efficiency of surface-bound fluorophore Bioderived Nanosupports environmentally friendly contaminant removal biocompatible disinfection programmable structure that allows enhanced enzyme selectivity and activity biocompatible contaminant removal enhanced overall activity of enzyme couples

bacteriophage P22 capsid,29 bacteriophage Qβ,30 cowpea chlorotic mottle virus,31 and filamentous potato virus X.32 Enzymes can be immobilized on these particles by genetic or chemical fusion to the capsid protein or a binding tag. Molecular biotechnologies provide a comprehensive toolbox to manipulate protein structures, which empowers rational modification of protein cages to improve enzyme affinity and specificity. Genetic functionalization of protein units in assembled cages alters their surface charges and hydrophobicity and adds chemical recognition sites, allowing customization of protein cages to adsorb or repel specific contaminants. The use of such programmable protein cages could enhance the enzyme selectivity and improve the removal efficiency of contaminants at trace levels. 2.4.2. DNA Origami. Complementary Watson−Crick base pairing between adenine and thymine and between guanine and cytosine maintains the DNA double-stranded structure in nature. This high specificity of base pairing is used to assemble DNA nanocompartments, named origami, from singlestranded DNA sequences with complementary overlaps in vitro.33 Enzymes are immobilized on DNA origami via interactions between the DNA motifs on enzyme molecules and the complementary motifs on DNA supports.33,34 As the structures of DNA origamis and their interactions with enzyme molecules are rationally designed, spatial arrangement and positioning of enzyme molecules on DNA surfaces can be predicted and precisely controlled.34 This benefits the enzymatic catalysis by boosting enzyme activities through the creation of substrate channeling.34 As summarized in Table 1, each of the above-discussed nanomaterials offers unique benefits as an immobilization support. Before we begin to discuss potential applications of nanosupported enzymes in water supply systems, it is worth noting that direct determination of the optimum nanosupport based on published studies may be difficult because different enzymes respond differently to a nanomaterial.35,36 For example, zinc-MOF-immobilized cytochrome c showed a 10-

14

10 20, 54, 56

23, 26, 29, 37, 41

33, 34

fold increase in activity compared with free cytochrome c, while the activity of horseradish peroxidase in zinc-MOF was about 4 times lower than that of the free enzyme.16 Therefore, for solving particular water issues, the selection of enzyme is crucial, and comparison of different nanomaterials in laboratory studies is indispensable to determine the most suitable nanosupport.

3. APPLICATIONS OF NANOSUPPORTED ENZYMES IN WATER PURIFICATION AND MONITORING 3.1. Applications in Water Treatment

3.1.1. Removal of Emerging Contaminants. Emerging contaminants are ubiquitous in drinking water and often difficult to remove in conventional water treatment processes such as coagulation, sedimentation, and filtration. To improve the removal efficiency of these chemicals, many advanced techniques have been developed and employed, including photocatalysis, electrochemical oxidation, enzymatic catalysis, and advanced chemical oxidation. Among them, the enzymatic method shows relatively high efficiency and versatility, requires less chemical and energy usage, and has a lower tendency to generate toxic byproducts, which collectively make it a suitable candidate particularly for POU systems.4,6,37 To date, many enzymes such as peroxidases, laccase, organophosphate hydrolase, and atrazine chlorohydrolase have been identified and designed to treat various contaminants, including endocrine disruptors, pharmaceuticals, pesticides, and personal care products. Mechanisms, kinetics, and pathways of enzymatic treatment have been extensively reviewed elsewhere.4,35,36 One of the challenges of applying enzymatic approaches is that free enzymes could be quickly inactivated because they are susceptible to ligands and metal ions in natural waters and even products from their own activities. This challenge can be overcome by immobilizing enzymes on nanosupports, which generally improves their stability and extends their functional life with minimal loss of activity.14 D

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glucose oxidase and copper nanoflowers with immobilized HRP. However, this nanoflower approach relies on random encapsulation events, which does not allow systematic control of the distance between enzymes. In this regard, DNA origami provides significant advantages because the localization of immobilized enzymes can be precisely controlled using spatially addressable DNA scaffolds, further promoting the activity of enzyme couples.34 Although most of the reported studies have focused on batch experiments, continuous flow systems in which enzyme− nanomaterial composites are retained and reused are probably more practical and cost-effective in real-world POU systems.35 One potential solution is to pack beads bearing immobilized enzyme−nanomaterial composites into columns for use in fluidized bed reactors. Alternatively, nanosupported enzymes can be used in dispersed form and held in membrane reactors using ultrafiltration membranes.38 Such column/membrane continuous reactors can be used as an easy “add-on” to existing POU water treatment systems to improve their efficiency toward contaminant removal. 3.1.2. Disinfection. Current drinking water disinfection processes such as chlorination and ozonation can produce harmful byproducts. In contrast, antimicrobial enzymes limit production of any harmful byproducts, as they fight microbes and bacterial biofilms by interrupting their cellular components or adhesion.7 Lysozyme and lysostaphin are efficient enzymes against Gram-positive bacteria because they cleave crosslinking bonds found in peptidoglycans, a major component of the Gram-positive cell wall.43,44 An alternative approach is to use enzymes that generate oxidative stressors like hydrogen peroxide to suppresses all bacterial growth.29 Proteases can control microbial fouling by hydrolyzing adhesion proteins essential for bacterial attachment to the surface or to other bacteria.7 Other examples of antimicrobial enzymes include lysosomal extract, subtilisin, and bacteriophage lysin, as summarized elsewhere.7,45 Immobilization of antimicrobial enzymes improves their stability and prevents them from being digested by microbes, which facilitates their implementation in continuous column reactors that can be used as separated disinfection units and their incorporation in other POU processes to control biofouling.44 Moreover, conjugation to nanosupports preserves enzyme activity during further manipulations, which can expand applications of antimicrobial enzyme−nanomaterial composites. For instance, CNT− protease conjugates were incorporated in a polymer matrix to prepare highly active and stable antifouling films, which may be used as coatings on inner surfaces of water storage containers to reduce microbial fouling and improve water storage safety.46

Immobilization also allows recycling and repeated use of enzymes, which lowers the overall cost. In a pilot study, a membrane reactor loaded with laccase immobilized on silica nanoparticles removed approximately 66% of the feed bisphenol A from wastewater flowing at 78 L/h during a 45day test, and this treatment had similar cost to ozonation and activated carbon sorption.38 Metallic nanosupports have been demonstrated to enhance the activity of immobilized enzymes.17,18,39 Although the exact mechanisms are still not fully understood, there is evidence that increased metal ion concentrations in the microenvironment of the metallic nanosupports and their cooperative effect with immobilized enzymes contribute to the increased activity. For instance, immobilization of 2,4-dichlorophenol hydroxylase in copper nanoflowers increased degradation of 2,4dichlorophenol up to 160%.39 The enhancement effect is more significant for enzymes with cofactors that contain metal ions. A study of horseradish peroxidase (HRP), a heme-containing enzyme, showed that both HRP−iron nanoflowers and HRP− copper nanoflowers were at least 3 times more active and stable than free HRP.18 Interestingly, the HRP−iron nanoflower had better activity and stability than the copper nanoflower, which could be attributed to the synergistic effects between the iron nanoflower and heme iron in the enzyme. Thus, immobilization of enzymes on metallic nanosupports comprising the same metal species as the enzymes might offer the highest benefits. Treatment of contaminants at trace levels presents a difficult challenge for nearly all technologies because of slow removal kinetics. Nanosupports can adsorb and concentrate contaminants near the surface and in the microenvironment surrounding enzymes, thereby increasing the apparent enzyme kinetics and benefiting removal of contaminants. Fullerenes, CNTs, and graphene constitute a class of carbon nanomaterials that can effectively adsorb many organic contaminants.5 Their surfaces can be functionalized through chemical grafting to increase the selectivity toward specific contaminants. However, such modification could impact the activity of immobilized enzymes.40 Protein nanocages stand as another class of nanosupports with customizable contaminant targeting properties. A lumazine synthase protein cage was constructed with a supernegatively charged inner surface that preferentially captured positively charged substrates over negative and neutral substrates and inverted the substrate specificity of the encapsulated enzyme by 480-fold.26 By genetic attachment of an amphipathic peptide to the N-terminus of the major vault protein, vault nanoparticles with lipophilic cores were constructed.41 The modified vaults showed strong affinity to hydrophobic compounds and concentrated them inside. These engineered protein cages provide promising enzyme immobilization platforms for removal of charged and hydrophobic contaminants at trace levels. Immobilization of enzymes on nanosupports also provides advantages for using enzyme couples. Removal and detoxification of certain contaminants (e.g., trichloropropane) require step-by-step conversion using multiple enzymes,42 whose efficiency is partially limited by substrate diffusion between different enzymes. Colocalization of the enzymes on small surfaces of nanosupports could shorten the internal diffusion route, benefiting the overall kinetics. Glucose oxidase and HRP coembedded on copper nanoflowers showed 5 times higher activity than free enzymes and 60% higher response than a mixture of copper nanoflowers with immobilized

3.2. Applications in Water Monitoring

Traditional manual methods to monitor contaminants are expensive and time-consuming, and a lack of timely detection can increase health risks. Thus, the development of sensors for rapid on-site detection of contaminants can greatly lower the risk of incidental or accidental exposure to communities and also provide guidelines for designing POU systems to meet specific treatment demands. Because enzymes have the advantages of high sensitivity, high specificity, and rapid response to substrates, they represent a promising approach for the development of biosensors. Nanosupports provide the structure needed to develop enzyme-based sensors. E

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Accounts of Chemical Research 3.2.1. Contaminant Monitoring: Electroenzymatic Approach. Electroenzymatic biosensors consist of enzymes coated on electrodes and are often used in detection of contaminants. The species generated or consumed by enzymatic activity produce current when potential is applied, and the activity depends on the contaminant levels. However, challenges that could limit the performance of this type of sensor include loss of activity and stability of enzymes and electron transfer barriers created by enzyme coatings on the electrode surfaces.47 In this regard, conjugation of enzymes to conductible nanosupports such as carbon nanomaterials (e.g., CNTs, graphene) and nanometals (e.g., gold nanoparticles) increase the stability and loading of enzymes and promote electron transfer on electrodes, thereby yielding sensitive and rapid-response sensors.5 Depending on whether contaminants react with the enzyme or indirectly affect the enzyme activity, two main detection strategies have been developed. First, species generated by enzymatic catalysis of contaminants can be directly detected amperometrically (Figure 2A). By the use of an electrode

enzymatic sensors, they are less sensitive but more convenient for use by the nontrained public. The generation of colored products from enzyme-mediated contaminant conversion can be directly used to quantify concentrations. Conjugation to nanosupports helps improve enzyme stability and activity, leading to lower detection limits and faster responses, and facilitates easy transfer of the methodology from laboratory settings to field applications.17,53 The unique surface and optical properties of noble metal nanomaterials also play important roles in developing colorimetric enzyme sensors. Nanometals are able to enhance the quantum efficiencies of surface-bound fluorophores. A fluorophore that is a competitive enzyme inhibitor can bind to the immobilized enzyme and stay on the surface of the nanometal particle, thus exhibiting enhanced fluorescence (Figure 3A). In the presence of the analyte, the fluorophore is displaced from the binding site and diffuses away from the nanometal, resulting in fluorescence decay.54 An alternative means of designing colorimetric sensors involves the distancedependent optical properties of nanometal particles, which has been mainly explored for detecting heavy metals (Figure 3B).20 The principle is that the aggregation state of the nanometal is controlled by the contaminant-dependent enzyme activity. DNA/RNAzymes are nucleic acid strands that act as ribonucleases to recognize and cleave nucleic acid fragments at specific sites, and they have high specificity and activity toward metal ions such as Pb2+ and Cu2+. The sensing relies on modification of nanometal particles with two nucleic acid strands: the first strand is the DNA/RNAzyme, and the second strand contains the recognition site and bridges nanometal particles to form agglomerates. In the presence of activator metal ions, the enzyme is activated and cleaves the bridge strand, causing disaggregation of nanometal agglomerates into smaller particles, which is reflected through a color output. As DNA/RNAzymes have high activity and specificity toward their activator ions, this approach is able to detect heavy metals in the submicromolar range and has high selectivity over many interfering metal ions.20 The affinity between the DNA/ RNAzyme and metal ions depends on its nucleic acid sequence and can be adjusted by introducing mutations.55 Thus, an important feature of the sensor is that the detection range can be tuned over several orders of magnitude by using enzyme mutants.20 3.2.3. Bacterial Monitoring. For use in bacterial detection, the nanosupport interacts with the cell surface while the immobilized enzyme generates the signal. βGalactosidase catalyzes the hydrolysis of chlorophenol red βD-galactopyranoside (CPRG), a chromogenic reaction that results in a color change. Gold nanoparticles modified with cationic ligands adsorb the anionic β-galactosidase and inhibit its activity. Upon the binding of anionic bacteria surface to the nanoparticles, immobilized β-galactosidase is displaced, restoring its activity to catalyze reactions that generate colorimetric output in the presence of CPRG. A test strip based on immobilized β-galactosidase has been successfully developed to detect bacteria at levels as low as 104 bacteria/mL.56 However, this method cannot differentiate pathogens from nonpathogenic strains, as it reacts with all anionic surfaces. Using enzymes that target specific extracellular substances or cellular components of pathogenic bacteria should improve the specificity.

Figure 2. Schematic of electrochemical enzymatic sensors. (A) The target contaminant is directly converted by the enzyme. The generated product is electrochemically converted, yielding an amperometric response. (B) The target contaminant is non-enzymereactive but affects the enzyme-catalyzed mediator conversion, causing a change in current at the electrode.

coated with CNT-immobilized organophosphate hydrolase, paraoxon was detected at 0.15 μM, a >10-fold improvement compared with the free-enzyme-doped electrode.48 Other contaminants, such as phenol,49 bisphenol A,50 and fenitrothion,51 can also be detected with suitable nanosupported enzymes. Because of the high specificity of enzymes, this approach has high selectivity over interfering compounds. Thus, contaminants can be detected at very low concentrations, even in mixtures. Second, inhibitory effects of nonenzyme-reactive contaminants on enzyme activities can be used to detect the contaminants (Figure 2B). The presence of the contaminant decreases enzyme-mediated reaction rates in a concentration-dependent manner, and the reduction in the reaction rate alters the current detected. Heavy-metal ions are inhibitors for many enzymes because they block thio groups of enzymes, forming mercaptides.8 An electrode coated with urease immobilized on gold nanoparticles was 2 times more sensitive against Hg2+ than the free enzyme electrode and was able to detect Hg2+ at levels as low as 50 nM.52 The inhibitory approach significantly expands the contaminant sensing range, but contradictorily, it is less selective and vulnerable to complex ingredients of natural water samples. 3.2.2. Contaminant Monitoring: Colorimetric Approach. Colorimetric approaches can be used for quick detection of contaminants levels. Compared with electroF

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Figure 3. Schematics of a colorimetric sensor based on unique optical properties of nanometals. (A) Surface-enhanced quantum efficiency of fluorophores on nanometals. The fluorophore binds to the surface-immobilized enzyme and shows enhanced fluorescence. In the presence of the targeted contaminant, the bonded fluorophore is displaced and diffuses away from the nanometal particles, leading to a decrease in fluorescence. (B) Aggregation-state-dependent optical properties of nanogold particles. The presence of Pb2+ activates DNAzyme 17E, which leads to disaggregation of nanogold agglomerates and further color change. Reproduced from ref 20. Copyright 2003 American Chemical Society.

Figure 4. Analysis of the feasibility of nanosupported enzymes in POU water supply systems: potential applications, limitations, and future directions.

4. LIMITATIONS Nanosupported enzymes have many advantages over free enzymes and offer great potential in providing clean water. However, challenges and gaps in their successful integration into POU water supply systems exist (Figure 4). The application of immobilized enzymes relies on their specificity and activity toward the contaminants. Although a number of enzymes have been identified and isolated, most of them act only on the same groups of contaminants, including phenolics, organophosphates, heavy metals, or certain bacterial species.4,8 For other contaminants, such as per- and polyfluoroalkyl substances, efficient enzymatic approaches are missing because of the lack of suitable enzymes. Meanwhile, current laboratorybased research mainly focuses on immobilizing single enzymes

on nanosupports. In view of the broad range of water contaminants and the fact that contaminant destruction pathways can require multiple enzymes,42 these single-enzyme systems are not yet efficient in dealing with complex issues in real-world applications. Moreover, certain nanomaterials used as supports could pose health risks, depending on the toxicity of the nanomaterial and the pathway of exposure. To date, many studies have demonstrated the cytotoxicity and ecotoxicity of nanomaterials and investigated their fate and transport in the environment.57 However, there are limited data available on their fate in engineered treatment processes, particularly in drinking water treatment and POU systems. Putting nanosupported enzymes into water treatment applications before their potential risks are fully understood G

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Accounts of Chemical Research Biographies

might cause unintended detrimental consequences. Lastly, current costs of nanomaterials and enzymes are high,4,5 limiting access to them in less prosperous regions, where the highest demands for POU treatment are present. However, with rapid advances in biotechnology and maturation of technology, their cost is expected to decrease.

Meng Wang is a postdoctoral scholar at UCLA. He received his B.S. in Environmental Science from Nanjing University in 2012 and his M.S. in 2015 and Ph.D. in 2018 in Civil and Environmental Engineering from UCLA under the supervision of Shaily Mahendra. His research focuses on developing bionanomaterial-immobilized enzymes for water treatment applications.

5. CONCLUSIONS AND FUTURE PERSPECTIVES Nanosupported enzymes are promising technologies to improve POU water supply systems. Immobilized biodegradative enzymes have the potential to develop effective, ecofriendly, portable water treatment technologies, and they offer a versatile platform in which the enzyme and the nanomaterial can be customized to provide on-demand treatment. Nanosupported antimicrobial enzymes provide a safe disinfection alternative and can also improve water storage safety. Highperformance sensors based on nanosupported enzymes enable rapid on-site diagnosis of contamination, presenting the basis for determining specific treatment demands and designing suitable POU sets. However, challenges associated with versatility, potential health risks, and cost exist, hindering the technology transfer from laboratory settings to field POU systems. To accelerate the use of nanosupported enzymes in POU technologies, we propose the following four recommendations for future studies (Figure 4). First, there is a need to explore many more enzymes to extend their applicability in contaminant removal. The techniques include identification and isolation from natural biological processes as well as rational design and directed evolution. Second, multienzyme immobilization systems should be developed to deal with complex issues in field applications. In this regard, bioderived materials provide some potential advantages over others supports because their programmable structures enable precise control of enzymes’ spatial placement, promoting the activities of coupled enzymes.34 Third, studies should evaluate whether conjugation to enzymes would mitigate the toxicity of nanosupports because coating and functionalization have been shown to alter nanomaterials’ behavior and toxicity.57 If not, efforts should be made to investigate the fate and transport of nanosupported enzymes in POU systems and strategies to minimize unintended releases of nanomaterials. Finally, the use of purified enzymes in immobilization requires significant expense. Some nanosupports, such as the vault nanoparticles, have been shown to immobilize enzymes through specific interactions.22 They are able to selectively capture targeted enzymes, eliminating the need for enzyme purification. Developing such multifunctional nanosupports that simultaneously purify and immobilize enzymes will provide economic benefits to the nanosupported enzyme systems.

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Sanjay K. Mohanty is an Assistant Professor in the Department of Civil and Environmental Engineering at UCLA. He earned his Ph.D. in 2011 from the University of Colorado, Boulder. His research aims to understand the links between weather conditions, subsurface contaminant removal processes, and water quality impairments during climate change and to develop sustainable engineering solutions to increase the resilience of stormwater infrastructure, low-cost water treatment, and soil remediation technologies. Shaily Mahendra is an Associate Professor and Samueli Fellow in the Department of Civil and Environmental Engineering at UCLA. She received her Ph.D. in 2007 from the University of California, Berkeley. Her research areas are microbial processes in natural and engineered systems, applications of molecular and isotopic tools in environmental microbiology, environmental implications and applications of nanomaterials, and bioremediation of emerging water contaminants.

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ACKNOWLEDGMENTS

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REFERENCES

We acknowledge support from 2017 Paul L. Busch Award from the Water Research Foundation to S. Mahendra.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Meng Wang: 0000-0002-8430-660X Shaily Mahendra: 0000-0003-3298-9602 Notes

The authors declare no competing financial interest. H

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