Catalytic Biosensors from Complex Coacervate Core Micelle (C3M

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Catalytic Biosensors from Complex Coacervate Core Micelle (C3M) Thin Films Hursh V. Sureka,† Allie C. Obermeyer,‡ Romeo J. Flores,† and Bradley D. Olsen*,† †

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States Department of Chemical Engineering, Columbia University, New York, New York 10027, United States

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ABSTRACT: Enzymes have been applied to a variety of industrially and medically relevant chemistries as both catalysts and sensors. Incorporation of proteins and enzymes into complex coacervates has been demonstrated to improve the thermal, chemical, and temporal stability of enzymes in solution. In this work, a neutral-cationic block copolymer and an enzyme, alkaline phosphatase, are incorporated into complex coacervate core micelles (C3Ms) and coated onto a solid substrate to create a biocatalytic film from aqueous solution. The incorporation of photo-cross-linkable groups into the neutral block of the polymer allows the film to be crosslinked under ultraviolet light, rendering it insoluble. The morphology of the film is shown to depend most strongly on the protein loading within the film, while solvent annealing is shown to have a minimal effect. These films are then demonstrated as specific sensors for Zn2+ in solution in the presence of other metals, a model reaction for ion-selective heavy metal biosensing useful in environmental monitoring. They are shown to have low leaching and maintain sufficient activity and response for sensing for 1 month after aging under ambient conditions and at 40 °C and 50% relative humidity. The C3M immobilization method demonstrated can be applied to a wide variety of proteins with minimal chemical or genetic modification and could be used for immobilization of charged macromolecules in general to produce a wide variety of thin-film devices. KEYWORDS: biosensors, coacervation, block copolymers, enzyme immobilization

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INTRODUCTION Enzymes are green, biodegradable catalysts that are typically selective, sensitive, and highly enantio- and stereo-specific. They have been applied to a variety of industrially and medically relevant chemistries including glucose isomerization,1,2 pharmaceutical synthesis,3−6 biodiesel production,7,8 and carbon dioxide sequestration.9,10 Enzymes are most readily used as homogeneous catalysts, but heterogeneous catalysts are desirable in both biocatalysis and sensing applications because they allow for longer usage and easier purification without loss of the catalyst.1,2,6−12 Enzyme immobilization presents a promising route to stabilize proteins against chemical and thermal stress, maximizing the useful lifetime of a proteinbased device.1,2,6−14 Surface immobilization is the most commonly used enzyme immobilization method, and it has been applied to make both biocatalysts and biosensors. Notable examples include glucose and ethanol sensors15 and ELISA assays,16 along with some industrially available glucose isomerase1,2 and lipase products.17 Surface immobilization can be achieved by either passively binding the protein to a substrate through van der Waals forces, electrostatic forces, etc. or by covalently binding the protein to a functionalized surface.9,11,15,18 Despite its common usage, surface immobilization is known to have low protein loading and reduced function due to protein orientation defects.15,16 The use of spacers is thought to © XXXX American Chemical Society

allow for greater translational and conformational freedom to try to overcome the issue of protein orientation defects.15 To increase the shelf life of these sensors and catalysts, proteins can be cross-linked with glutaraldehyde or supplemented with polyelectrolytes or polyols.9,11,15,18 Alternative immobilization methods try to increase protein loading and protein mobility by moving from a twodimensional surface to a three-dimensional matrix. Entrapment, encapsulation, layer-by-layer (LbL) assembly, complex coacervation, and protein−polymer block copolymer selfassembly are methods that have been demonstrated to create three-dimensional enzymatic systems.9,19−26 Among the many different methods, the use of LbL technologies and complex coacervates to achieve immobilization have garnered significant attention in recent years due to their relative ease.18,21,23−30 Complex coacervate-based methods enable coating to be achieved in only one step, which is attractive for manufacturing.19,23,25,27,30 Both complex coacervates and complex coacervate core micelles (C3Ms) have been demonstrated to achieve high protein loading by mass and to improve the stability of encapsulated proteins to temporal, thermal, and chemical Received: May 15, 2019 Accepted: June 26, 2019

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DOI: 10.1021/acsami.9b08478 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Synthesis of (POEGMA-r-BP)-b-qP4VP via RAFT polymerization. POEGMA-r-BP was first polymerized via RAFT, and then, the polymer was used as a macroCTA to polymerize (POEGMA-r-BP)-b-P4VP. (POEGMA-r-BP)-b-P4VP was then quaternized with methyl iodide.

degradation.21,26,27,30−33 Encapsulation of GFP, lipase, organophosphate hydrolase, and many other proteins has been demonstrated.21,26,27,30−33 Supercharging of proteins has been demonstrated to aid in the incorporation of proteins into both complex coacervates and C3Ms and has allowed for proteins to replace one of the polyelectrolytes in the system.25,34,35 The formation of both phases is well predicted by the negative charge ratio of the protein when combined with a strong polyelectrolyte.25 Thus, enzymes can be genetically engineered20 or chemically modified to form complex coacervates and C3Ms.25,34,35 In cases where protein modification is difficult, complex coacervates and C3Ms incorporating enzymes can still be formed; however, the overall protein loading is lower.27 This is achieved by combining an anionic polymer, a cationic polymer, and a protein in solution and allowing the protein to partition into the complex coacervate or C3M phase.26,27,30 Charged-neutral diblock copolymers, like those used in C3Ms, have also demonstrated microphase separation, 36,37 and templating of mCherry has been demonstrated in C3M films previously.19 The current standard for heavy metals analysis in fresh water is axially viewed inductively coupled plasma-atomic emission spectrometry, per the U.S. Environmental Protection Agency (EPA).38 This technology requires a trained technician to operate, a clean workspace, and electricity. Technologies are under development to overcome these limitations. One household test that is available today uses a color-changing paper strip to estimate the concentration of heavy metals in water.39 While this works for civilian applications in the home, military and industrials applications, like detection of nuclear production and pollution levels, require specificity and a higher degree of precision. Several amperometric, field-effect transistor-based, and optical sensors have been developed.40 Förster resonance energy transfer has been leveraged to develop a Hg2+ sensor, where the presence of the ions causes the formation of a thymine−Hg2+−thymine hairpin structure that brings a quencher into close proximity of an organic fluorophore, diminishing its signal.41 Newer devices use more stable species like quantum dots for fluorescence and Au nanoparticles (NPs) to achieve a similar effect and nanometal surface energy

transfer, with a lower likelihood of photobleaching.42 Functionalized Au NPs have also been used to detect heavy metals colorimetrically, through a shift in the surface plasmonic resonance peak upon aggregation induced by the presence of the metals.40,43−45 Amperometric devices utilizing carbon nanotubes have also been developed with high sensitivity and specificity; however, they show poor performance after day-to-day use.40 The use of enzymes to accomplish heavy metal sensing is also under development. Satoh and co-workers have developed a flow sensor for Cu2+, Zn2+, and Co2+ using ascorbate oxidase and alkaline phosphatase (PhoA) based on apoenzyme reactivation.14 Urease, glucose oxidase, and horseradish peroxidase have been used for amperometric detection of heavy metals.46 A colorimetric assay for the detection of several different heavy metals using β-galactosidase deactivation on a paper strip has also been developed.47 This work demonstrates a method to create insoluble, biocatalytic films using a one-step aqueous coating process based on complex coacervation followed by photo-crosslinking chemistry. The enzyme alkaline phosphatase is used as a model protein for selective sensing of transition metals in water. It is genetically engineered to increase negative charge density and drive C3M formation with a photo-cross-linkable neutral-cationic block copolymer. The effects of film thickness, annealing conditions, and protein loading on film activity and morphology are evaluated with fluorimetric assays, scanning probe microscopy (SPM), and grazing-incidence small-angle X-ray scattering (GISAXS). The films are then tested for sensitivity and selectivity for Zn2+ under a variety of conditions including in the presence of various mixtures of metals and in river water. Finally, the films are tested for their thermal stability. The results demonstrate the viability of this immobilization method for the fabrication of a multitude of enzyme and macromolecule-based thin-film devices.

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RESULTS AND DISCUSSION Material Design. A polymer encapsulant for thin-film formation was designed using a neutral-cationic block copolymer synthesized by reversible addition-fragmentation chain transfer polymerization (RAFT). The synthesis of B


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Figure 2. (a) Structure of alkaline phosphatase (PDB: 3TG0)51 with mutations highlighted. (b) Table of mutants and corresponding mutations and α values. (c) kcat and (d) KM data for supercharged PhoA mutants determined by the fluorimetric assay measuring conversion of 4-MUP to 4MU. All mutants showed significantly greater kcat than that of the wild-type and a slight increase in KM. Mutant 4 was chosen for subsequent studies because it had sufficient negative charge ratio to enable complexation without a relative drop-off in activity.

Figure 3. (a) Particle mass distributions determined by DLS for mixtures of PhoA and (POEGMA-r-BP)-b-qP4VP. Complexes began to form at 5% (wt/wt), with micellization occurring at 40 and 50%, and the formation of larger complexes occurring past this point. (b) Effect of polymer addition on the activity of PhoA in solution. The polymer was added at 50% (wt/wt) with enzyme concentration remaining constant between the two cases. The effective kcat of the enzyme was reduced from 6.31 ± 0.01 to 3.84 ± 0.01 s−1 upon addition of the polymer, and the effective KM was found to decrease from 1.27 ± 0.03 to 0.94 ± 0.07 μM.

degrees of polymerization (DPs) of 74 and 67 for OEGMA/ BP and 4VP blocks, respectively, and dispersity (Đ) of 1.11 as confirmed by 1H NMR and size-exclusion chromatography (Figures S1−S5). The OEGMA/BP block was 9 mol % BP monomer. To prepare metal sensing catalysts, alkaline phosphatase (PhoA) was selected as a model enzyme. This enzyme can be used as a zinc sensor,14 a phosphate scavenger for bone scaffolds,13 and a detector for pesticides.49 It is known to be relatively easy to engineer and express in large quantities.50 To promote strong aggregation between the cationic polymer and protein, PhoA was genetically modified to have increased anionic charge. Previous work has shown that a negative charge ratio (α) of approximately 1.4 or greater is necessary for the formation of C3Ms,25 so the modifications were designed to surpass this level. Wild-type PhoA has α of 1.26. Genetic

poly[(oligo(ethylene glycol) methacrylate)-r-(benzophenone methacrylate)]-b-(methyl-quaternized 4-vinylpyridine)] ((POEGMA-r-BP)-b-qP4VP) was performed according to Figure 1. This polymer is advantageous for enzyme encapsulation because proteins are weakly interacting with the uncharged POEGMA block,25,27,48 the POEGMA and qP4VP are sufficiently segregated to yield ordered nanostructures in the absence of protein,36,37 and the rubbery-glassy contrast between the two blocks of the copolymer provides imaging contrast in SPM. First, the random copolymer of OEGMA and BP was synthesized via RAFT. The resulting polymer was then used as a macrochain transfer agent (macroCTA) in the polymerization of 4VP. The final product was quaternized by a reaction with iodomethane. The benzophenone concentration was optimized to achieve successful photo-cross-linking while also maintaining water solubility. The final polymer had C


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Figure 4. Method for immobilization of enzymes in C3M thin films. The polymer and protein are premixed to form complex coacervate core micelles and then flow coated onto a solid substrate. UV irradiation is used to cross-link the soft block via benzophenone groups, rendering the final film insoluble.

Figure 5. (a) Film activity for films of 50 wt % enzyme at varying thickness. The activity increase with thickness is minimal beyond about 150 nm. (b) Relative response to 5 ppm Zn2+ as a function of film thickness. The relative response is the ratio of the film activity after deactivation with EDTA and reactivation with ZnSO4 to the native film activity. The relative response increase was minimal after 150 nm.

40% (Rh = 23.8 ± 9.6 nm) and 50% (Rh = 39.9 ± 9.8 nm). Past this point, at 60 and 75% protein, larger aggregates began to form. A solution at 50% protein was chosen for all further work as this mixture formed micelles and was the point of maximum coacervation with the qP4VP homopolymer as per the method described by Obermeyer et al.25 (Figure S7). Polymer addition caused a slight decrease in the effective activity of the enzyme (Figure 3b). Solutions of the enzyme alone and the enzyme and polymer together at a 50:50 ratio were prepared with equal concentrations of the enzyme. The effective kcat of the enzyme was reduced from 6.31 ± 0.01 to 3.84 ± 0.01 s−1 upon mixing, and the effective KM was found to decrease from 1.27 ± 0.03 to 0.94 ± 0.07 μM. This could be due to noncompetitive inhibition of the enzyme,52 denaturation, or the effect of the local environment on activity.15 Enzyme Immobilization and Sensing within C3M Films. Enzyme immobilization was achieved by flow coating a 15−20% (wt/wt) solution of POEGMA-BP-b-qP4VP and PhoA M4 (50:50) in water onto a poly(ethylene glycol) (PEG)-treated Si surface and cross-linking under ultraviolet light (254 nm) (Figure 4). The protein is expected to segregate strongly into the charged domains, as shown in Figure 4, per the work done by Kim et al.19 The PEG treatment of the Si surface was necessary to allow for adhesion and wetting of the films to the surface. The films were photo-cross-linked for 5

modification was chosen over chemical charge modification to provide a single, reproducible molecular species for coacervation experiments. Five mutants were expressed and purified by periplasmic extraction and fast protein liquid chromatography (FPLC) using anion exchange. The structure of the protein and a summary of the mutations are shown in Figure 2a,b. Polyacrylamide gel electrophoresis (PAGE) analysis of the purified mutants is shown in Figure S6a,b. The enzyme activity was fit to Michaelis−Menten kinetics, and the negative mutants were found to have higher kcat than that of the native enzyme in solution, with slightly increased KM (Figure 2). A representative fit is shown in Figure S6c. PhoA Mutant 4 (K92D + K96D + K352D + K353D) was chosen for the sensor because of its high negative charge ratio of 1.50, high expression yield, and high activity. C3M Formation and Activity. Upon mixture of the protein and the block copolymer, the two components formed complex coacervate core micelles (C3Ms). Micelle formation was confirmed by dynamic light scattering (DLS) as a function of the polymer−protein blending ratio (Figure 3a) at 1 mg mL−1 in water. Both the pure polymer and pure protein showed minimal aggregation. Upon addition of 5% (wt/wt) protein to the polymer, a small amount of complexation was seen as peaks began to form above 10 nm. The level of complexation steadily increased until micelles were formed at D


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Figure 6. SPM height and phase images and GISAXS scattering patterns for films with 0% (a−c), 10% (d−f), 25% (g−i), and 50% (j−l) (wt/wt) protein loading. Scale bars are 200 nm. Z-scales are 20 nm*, 40 nm†, 80 nm‡, and 10° for phase images. Scattering intensity is presented on a logarithmic scale. Film thicknesses were 117 ± 2, 77 ± 5, 105 ± 7, and 126 ± 3 nm for 0, 10, 25, and 50 wt % protein loading, respectively. The pure block copolymer structures into disordered micelles under the conditions tested. With the increasing protein concentration, this structure gives way to the formation of larger features.

Figure 7. (a) Film activity and (b) relative response to 5 ppm Zn2+ as a function of protein loading. 0 wt % protein film showed no activity, so relative response was undefined, and there was no absolute response to Zn2+. The relative response is the ratio of the film activity after deactivation with EDTA and reactivation with ZnSO4 to the native film activity. Both activity and response increased with increased protein loading. Film thicknesses were 117 ± 2, 120 ± 2, 77 ± 5, 105 ± 7, and 126 ± 3 nm for 0, 5, 10, 25, and 50 wt % protein loading, respectively.

assayed. The ratio of the film activity after exposure to the sample to the initial film activity is referred to as the relative response and is used to quantify the film response to the heavy metals. Increasing film thickness was found to increase the film activity and response; however, this effect diminished beyond a certain thickness (Figure 5). The activity increased among the 53, 101, and 143 nm films but did not increase between the 143 and 175 nm films. The relative response also showed an increasing trend among the 53, 101, and 143 nm films but did not increase between the 143 and 175 nm films. These phenomena could be the result of transport limitations. Protein loading was found to have a significant effect on the morphology, activity, and response of the C3M films. The polymer alone showed a disordered micelle phase in both scanning probe microscopy (SPM) and grazing-incidence small-angle X-ray scattering (GISAXS) (Figure 6a−c). The SPM images show that large structures of phase-separated

min and soaked overnight in buffer prior to use. Photo-crosslinking and soaking had little effect on the structure of the C3M films (Figure S8). Based on the film color, which went from light blue (ca. 150 nm) to pale yellow (ca. 450 nm) or magenta (ca. 600 nm) after immersion, the films swelled by approximately 300−400% in solution. The films were found to be hydrophilic based on the contact angle analysis (Table S1). Reconstitution of the PhoA apoenzyme in various transition metal solutions, followed by activity assays, provides a basis for the selective detection of low-concentration heavy metals using the coacervate thin films. The sensors can achieve high selectivity for zinc within metal mixtures because PhoA apoenzyme becomes active only when it is reconstituted with zinc. In a typical sensing cycle, the coacervate films are first converted to the apoenzyme form by treatment with ethylenediaminetetraacetic acid (EDTA) to chelate any metal. The films are then incubated for a short period in a desired metal solution, and subsequently, the film activity is E


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Figure 8. SPM height and phase images for as-cast films (a, b) and films annealed with water (c, d), acetone (e, f), DMSO (g, h), and DMF (i, j). Scale bars are 200 nm. Z-scales are 35 nm for height images and 5° for phase images. All films are 50 wt % protein. Film thicknesses were 89 ± 2, 88 ± 3, 91 ± 2, 80 ± 4, and 83 ± 3 nm for the as-cast, water-annealed, acetone-annealed, DMSO-annealed, and DMF-annealed films, respectively. Solvent annealing did not greatly affect the morphology of the films showing large complexes in all samples.

Figure 9. (a) Film activity and (b) relative response to 5 ppm Zn2+ after annealing with various solvents. All films are 50 wt % protein. Relative response is the ratio of the film activity after deactivation with EDTA and reactivation with ZnSO4 to the native film activity. Treatment with water had no effect on activity, but all other treatments led to a marked decrease in both activity and response. Film thicknesses were 89 ± 2, 88 ± 3, 91 ± 2, 80 ± 4, and 83 ± 3 nm for the as-cast, water-annealed, acetone-annealed, DMSO-annealed, and DMF-annealed films, respectively.

thickness was held relatively constant at 105−126 nm, apart from the 10% protein film because this solution had noticeably lower viscosity than that of the others, producing a thinner film. Solvent annealing in polar and aqueous solvents was explored as a method to control morphology in the coacervate films. Because of the electrostatic nature of the coacervate interactions and the need to maintain enzymatic activity, solvents with a known ability to solubilize salt and aqueousbased formulations were the focus of annealing studies. Films were annealed with water, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetone, and ethanol. Solvents were mixed at a 50:50 volume ratio with methoxypoly(ethylene glycol) (Mw = 350) to reduce solvent activity, as described by Stewart-Sloan et al.36 Under these conditions, a film swelling of 30−50% was observed for water and acetone and less than 10% for DMSO and DMF based on color change in the films. Annealing led to little change in the height of the films after drying. Under all conditions tested, the films appear to form large complexes, with all solvents causing no significant change except DMSO that caused shrinking of the complexes (Figure 8). Grazing-incidence small-angle X-ray scattering (GISAXS) confirmed that there was no significant difference between films annealed with various solvents; however, the feature sizes observed in SPM would not be accessible by GISAXS (Figure S11). Annealing with solvents other than

protein−polymer complexes begin to form at 5% (wt/wt) protein loading (Figure S9) and increase in abundance at 10% (Figure 6d−f) and 25% (Figure 6g−i), until they fully dominate the film structure at 50% (Figure 6j−l). The GISAXS patterns show a peak for the disordered micelles of the pure polymer that gradually weakens with the increased protein loading up to 25% protein loading. The disordered micelles of the block copolymer alone were not visible by SPM past 0% protein loading; however, the GISAXS data suggest that the subsurface structure is still partially similar to that of the pure polymer. At 50% protein loading, the peak in the GISAXS pattern no longer appears, suggesting that the larger structures fully dominate the film structure at this point. This was further confirmed by one-dimensional (1D) analysis of the GISAXS data (Figure S10), with the diminishing peak in both qy and qz. While the precise structure of the complexes in the films is unknown, the trend in complexation with protein loading is consistent with the DLS data in Figure 3a, which showed a gradual increase in structures with radii greater than 10 nm with the increased protein loading until only the large structures were detected at 40 and 50% protein loading. The activity of the films is proportional to the protein loading (Figure 7a), as expected. The relative response, which should remain constant with protein loading (apart for the film with 0% protein), was maximized at the highest protein loading studied, which was 50% protein (Figure 7b). The film F


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Figure 10. (a) Relative response of C3M films to treatments with various M2+ at 5 ppm. The relative response is the ratio of the film activity after deactivation with EDTA and reactivation with M2+ to the native film activity. The average film thickness was 124 ± 16 nm, and all films were 50 wt % protein. Films showed high selectivity for zinc in all cases. The addition of other metals led to a decreased response to Zn2+. (b) Relative response of the C3M film to varying Zn2+ concentrations. The average film thickness was 97 ± 7 nm. The response was nonlinear after 10 ppb Zn2+ and constant after 50 ppb.

water led to a decrease in both the film activity (Figure 9a) and the relative response of the films (Figure 9b). Based on these results, water annealing was utilized for subsequent studies. When tested against a panel of various different metals individually, PhoA M4 C3M thin films were capable of sensing zinc specifically when compared to Cu2+, Co2+, and Ni2+ (Figure 10a), none of which showed a signal above the background. However, in the presence of a multimetal mixture, the overall response to zinc was reduced. Likely, this is due to competitive binding of the catalytic site by transition metals that are not catalytically active. Additionally, the presence of biological contaminants was found to have no statistically significant effect on the response of the films (Figure S12). When tested with Zn2+ alone, the response followed Langmuir isotherm behavior (Figure 10b) and was fit to a scaled Langmuir isotherm c

R(c M2+) = (R max − R min) ×

Figure 11. C3M films were tested under a variety of conditions, and the samples were doped with 10 and 50 ppb Zn2+. This included a control in Millipore water; 10 ppb Cu2+, Co2+, and Ni2+; Charles River water (CRW); filtered Charles River water (CRWF); and two mixtures of metals (Mix 1, 5 ppb Cu2+, 1 ppb Co2+, 4 ppb Ni2+, and 7 ppb Zn2+; Mix 2, 2 ppb Cu2+, 6 ppb Co2+, 2 ppb Ni2+, and 4 ppb Zn2+). The response to 50 ppb Zn2+ was found to be statistically significant in all cases (**p < 0.01, *p < 0.05), except in CRWF (p = 0.052). The p-values are summarized in Table S2. Films had an average thickness of 128 ± 16 nm, and all films were 50 wt % protein.

2+

K MM 2+ M

c

2+

1 + K MM 2+ M

+ R min (1)

where R is the relative response, Rmax is the relative response at 5000 ppb Zn2+, Rmin is the relative response at 0 ppb Zn2+, cM2+ is the concentration of the metal in solution in ppb (μg L−1), MM2+ is the molecular mass of the metal (g mol−1), and K is the equilibrium constant in μM−1. The equilibrium constant, K, was found to be 8.70 μM−1. This behavior is consistent with a set number of binding sites within the film, and the active site of the enzyme is expected to either be empty or have a bound metal center. In the absence of contaminants, the limit of detection was found to be less than 5 ppb Zn2+. The films were tested under a variety of conditions, including addition of contaminants, in water drawn from the Charles River before (CRW) and after filtering (CRWF), and in mixtures of metals (Figure 11). The samples were doped with 10 and 50 ppb of Zn2+. The two metal mixtures tested were 5 ppb Cu2+, 1 ppb Co2+, 4 ppb Ni2+, and 7 ppb Zn2+ (Mix 1) and 2 ppb Cu2+, 6 ppb Co2+, 2 ppb Ni2+, and 4 ppb Zn2+ (Mix 2). The samples in Charles River water and in the two mixtures had a higher baseline response due to contaminants in the river water and the basal level of Zn2+ in the mixtures, but in all cases, the response increased with increasing Zn2+ concentration. The response to 50 ppb Zn2+ was statistically significant compared to the control (p < 0.05) in all cases,

except in filtered Charles River water (p = 0.052), which has a somewhat lower mean value at 50 ppb and higher variance than other samples. In the control, 10 ppb Cu2+, and 10 ppb Co2+ samples, there was a statistically significant response to 10 ppb Zn2+. The p-values for all conditions are summarized in Table S1. Film Stability to Thermal Degradation. Alkaline phosphatase has been shown to have high thermal stability and can be held at temperatures higher than 80 °C for 5−30 min with minimal loss of activity in the presence of Mg2+, so the films were expected to have high thermal stability.53−55 Films were tested for thermal stability in three environments: ambient conditions (approximately 20 °C and 5−30% relative humidity (RH)), at 40 °C and 50% relative humidity (RH), and at 70 °C and 4% RH. The physical appearance of the films did not change over time. The activity of the films decreased by 30.1 ± 5.7% under ambient conditions (Figure 12a) and by 24.0 ± 7.2% at 40 °C and 50% RH (Figure 12b) over 30 days. This decay occurred mostly within the first 3 to 7 days, which G


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Figure 12. Activity and response of films aged under ambient conditions (a, d), at 40 °C and 50% RH (b, e), and at 70 °C and 4% RH (c, f). Films retained their activity and response under ambient conditions and at 40 °C and 50% RH, but were greatly inactivated at 70 °C and 4% RH leading to unreliable response. Film thicknesses were 111 ± 2, 122 ± 2, and 142 ± 1 nm for ambient conditions, 40 °C and 50% RH, and 70 °C and 4% RH, respectively.

may be due to some initial enzyme inactivation, the film drying out over time, or equilibrating with the environment, but the films seem to be stable past that point. The enzyme alone has been shown to have a similar activity decay profile at 20 °C in solution.56 The activity of the films at 70 °C and 4% RH decayed by 84.5 ± 20.6% over 30 days (Figure 12c). At 70 °C, the enzyme alone in solution was nearly fully deactivated after 3 days.56 This decay occurred rapidly with a 47.9 ± 9.6% decrease in activity happening in the first day. At both ambient conditions and 40 °C and 50% RH, the films retained sufficient activity to be usable. The relative response of the films under ambient conditions (Figure 12d) and 40 °C and 50% RH (Figure 12e) decayed by 29.5 ± 13.4 and 27.9 ± 5.8%, respectively, similar to the decay seen in activity. If the decrease in activity were purely due to enzyme deactivation, the relative response would be constant over time, since relative response is scaled by the activity of the film at the given time point. The response of the films aged at 70 °C and 4% RH (Figure 12f) initially decreased but became unreliable because of the decay in the film activity. The decay in response at both ambient conditions and 40 °C and 50% RH mostly occurred within the first 3 days to 1 week, after which there was an apparent steady state and there was sufficient activity for sensing after 1 month. This suggests that after a brief curing period, the films may have an extended shelf life.

caused by transport limitations or inhibition. These micelles were successfully cast into thin films, and the effects of film thickness, protein loading, and solvent annealing on morphology, activity, and response to Zn2+ were evaluated. The film thickness was found to have a diminishing effect on the activity and response of the films above a critical thickness. SPM and GISAXS studies of the films showed that protein loading had a significant effect on the morphology of the films as the underlying microphase structure of the block copolymer gave way to formation of larger complexes with increased protein loading. Both the activity of the films and the response of the films increased with protein loading. Solvent annealing was found to have little effect on the film morphology, but it caused both the activity and response to decrease for all solvents tested except water. The films were found to have both high selectivity and sensitivity to Zn2+. Selectivity was demonstrated in the presence of Cu2+, Co2+, and Ni2+, and the response to Zn2+ was found to follow a Langmuir isotherm. The response was tested under a variety of conditions and was found to be reliable for detection of 50 ppb Zn2+ in a range of aqueous samples. Aging under ambient conditions and 40 °C and 50% RH led to slight decreases in both the activity and response; however, aging under 70 °C and at 4% RH both led to a sharp decrease in the activity, leading to an unreliable response to Zn2+. The C3M film immobilization method can be easily employed on a range of enzymes with minimal genetic engineering or a simple chemical modification. When used with alkaline phosphatase, this technology allows for selective sensing of zinc, the metal native to PhoA and serves as a model for generalized enzymatic sensing of metals. This immobilization technique could be employed on a variety of enzymes and macromolecules, and by tuning the polymer chemistry, immobilization could be achieved on a variety of substrates,

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CONCLUSIONS Immobilization in C3M films has been demonstrated as an effective encapsulation and stabilization method for enzymes, and this work shows that this technology may be used to produce catalytically active polymer−protein hybrid thin films for biosensors. Micelle formation between charged block copolymers and proteins was confirmed via DLS and was found to maintain enzyme activity with a decrease in kcat likely H


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polymer was precipitated in diethyl ether and dried under vacuum. The degree of quaternization was >95% as determined by 1H NMR (400 MHz, D2O) based on the emergence of a methyl peak at δ 4.24 (s, 3 H) (Figure S3). Cloning of Charge Mutants of PhoA. The gene for PhoA in the pTrc99a plasmid was obtained as a generous gift from Dr Jeff Glasgow. The QuikChange II site-directed mutagenesis kit (Agilent) was used to mutate the pTrc99a-PhoA plasmid to generate five mutants of alkaline phosphatase: (M1) K92D + K96D, (M2) K92D + K93D + K96D, (M3) K352D + K353D, (M4) K92D + K96D + K352D + K353D, and (M5) K92D + K93D + K96D + K352D +K353D. The supplied protocol was followed.58 The optimal PhoA mutant was found to be Mutant 4. This mutant was used for all subsequent studies. The gene sequence and primer sequences are shown in Figures S15 and S16, respectively. Expression and Purification of PhoA. Engineered genes were transformed into Tuner cells following a standard protocol from Novagen. An overnight culture (5 mL) of the cells was prepared in LB media supplemented with 0.2 mg mL−1 ampicillin and grown at 37 °C. The overnight cultures were added to 1 L of 2xYT media supplemented with 0.2 mg mL−1 ampicillin and grown at 37 °C. When the optical density at 600 nm reached 0.4, the cultures were induced with 1 mM IPTG, 1 mM MgSO4, and 0.1 mM ZnSO4 and allowed to grow overnight at 37 °C. The cells were pelleted by centrifugation at 3500 × g for 10 min and resuspended in 50 mM Tris buffer, pH 8.0, after each centrifugation. The periplasm was isolated by adding solid sucrose (0.5 M), EDTA (2.5 mM), and lysozyme (0.6 mg mL−1) to the cells and incubating at 37 °C for 30 min. The sphaeroplasts were removed by centrifuging at 10 000 × g for 20 min. The resulting supernatant was the periplasmic fraction. The periplasmic fraction was precipitated by heating the solution for 10 min in an 80 °C water bath. The mixture was centrifuged at 24 000 × g to remove precipitates. The supernatant was dialyzed against 20 mM Tris, pH 8.0, and then purified via anion exchange chromatography using two 5 mL Q columns (HiTrap Q HP, GE Healthcare) in series equilibrated with 20 mM Tris, pH 8.0, and eluted with a 0−0.5 M NaCl gradient using the GE Akta FPLC. Fractions containing PhoA were identified by UV absorbance at 280 nm and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. These fractions were pooled and dialyzed against 20 mM Tris, pH 8.0, with 1 mM MgSO4 and 0.1 mM ZnSO4 and then stored at 4 °C. Dynamic Light Scattering (DLS). DLS was done on a Wyatt Möbius. PhoA was exchanged into water by ultrafiltration and diluted to 1 mg mL−1. (POEGMA-r-BP)-b-P4VP was dissolved in water at 1 mg mL−1 in water. Samples were then made by blending the two solutions at the appropriate ratios. Samples were allowed to equilibrate overnight at 4 °C. Each sample was measured three times for 10 s. Grafting PEG to Silicon Wafers. Silicon wafers (WaferWorld, (100) orientation) were cleaned with an air plasma for 1 min. An approximately 0.25 cm-thick layer of poly(ethylene glycol) monomethyl ether (PEG, Mn = 750) (Aldrich, #202495) was applied to the wafer. The coated wafer was then heated to 150 °C for 18 h under vacuum. Wafers were stored in acetone prior to use. Flow Coating of Coacervate Solutions. PEG-coated wafers were rinsed with acetone, methanol, and water and dried using filtered air. PhoA was exchanged into water four times, diluting by a factor of 10 each cycle, and concentrated to 15−20% (wt/wt) via ultrafiltration (Amicon Ultra 30K-15 mL). The concentration was measured via absorption at 280 nm. A 25% solution of POEGMA-BP-b-qP4VP was prepared in water and mixed with the protein solution to achieve a 50:50 mass ratio. The final mixture was diluted to 15−20%. The solution was then flow coated using a setup similar to that described by Stafford et al.59 onto the substrate under 50−70% RH at room temperature. Films were then annealed with water vapor in a chamber containing 0.6 M NaCl in water overnight and cross-linked under ultraviolet light for 5 min (UVP Model UVGL-15, 4 W, 254 nm), unless otherwise stated. Scanning Probe Microscopy (SPM). The NTEGRA system from NT-MDT was used to take height and phase scans using a

making this a general approach for biocatalytically active thinfilm fabrication.

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METHODS

Benzophenone Monomer Synthesis (BP). Benzophenone methacrylate was synthesized following a previously reported protocol.57 Triethylamine (10 mL) was added to a solution of 4hydroxybenzophenone (10 g, 50 mmol) in dichloromethane, and the solution was cooled to 0 °C in an ice water bath. A solution of methacroyl chloride (5.9 mL, 60 mmol) in dichloromethane was added dropwise while stirring. The reaction mixture was subsequently allowed to warm and stirred at room temperature overnight. The mixture was washed twice each with water, saturated NaHCO3, and brine. The organic layers were dried over Na2SO4, filtered, and the solvent was evaporated. The resulting residue was purified by automated flash chromatography using a Biotage system with dichloromethane and methanol as a mobile phase. 1H NMR (400 MHz, CDCl3): δ 7.90 (m, 2 H), 7.83 (m, 2 H), 7.62 (m, 1 H), 7.52 (m, 2 H), 7.29 (m, 2 H), 6.42 (m, 1 H), 5.84 (m, 1 H), 2.12 (m, 3 H) (Figure S13). LRMS (electrospray ionization, ESI) calculated for C17H15O3 ([M+H]+) 267.3, found 267.1 (Figure S14). POEGMA-b-qP4VP Synthesis. RAFT polymerization was used to synthesize a block copolymer from 4-vinylpyridine (4VP) (95%, Aldrich) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA, Mn = 300 g mol−1) (Aldrich) with a small fraction of a benzoylphenyl methacrylate (BP) with a narrow molecular weight distribution. OEGMA and 4VP were passed through basic alumina columns prior to polymerization to remove inhibitors. 4-Cyano-4(phenylcarbonothioylthio) pentanoic acid (Aldrich, 133 mg, 0.50 mmol) and azobisisobutyronitrile (AIBN) (recrystallized twice from methanol, 15.6 mg, 0.10 mmol) were added to a solution of OEGMA (20 g, 66.6 mmol) and BP (1 g, 3.8 mmol) in 60 g of 1,4-dioxane in a molar ratio of 140:8:1:0.2 (OEGMA/BP/CTA/initiator). The solution was degassed by three freeze−pump−thaw cycles. The polymerization was carried out in a sealed flask at 65 °C and terminated after 7.5 h by removal of heat and exposure to oxygen. The polymer was then precipitated in hexane and dried under vacuum. The POEGMA-r-BP copolymer was found to have Mn = 22.3 kg mol−1 and Đ = 1.13 (dn/dc = 0.056) as determined by size-exclusion chromatography (SEC) (Figure S4). SEC analyses were performed on a Waters high-performance liquid chromatography (HPLC) system equipped with a Waters 1515 Isocratic HPLC Pump with two columns (ResiPore, 300 × 7.5 mm, up to 500 kDa, Agilent Technologies, CA) in series. DMF with 0.02 M LiBr was used as the eluent with a flow rate of 1 mL min−1 at 70 °C. The detector system consisted of a Wyatt miniDAWN TREOS multiangle light scattering detector and a Wyatt Optilab T-rEX differential refractive index detector. The ratio of BP to OEGMA was determined by 1H NMR (400 MHz, CDCl3, Figure S1) by comparison of peaks at δ 7.52 (m, 3 H) and 7.84 (m, 4H) for BP and the 3.39 (s, 3 H) peak for OEGMA. The POEGMA-r-BP was then used as a macromolecular chain transfer agent for RAFT polymerization of 4VP. 4VP (3.68 g, 35 mmol) and AIBN (3.2 mg, 0.020 mmol) were added to a solution of the POEGMA-BP copolymer (3.24 g) in 12.5 g of a mixture of 1,4dioxane and DMF in a molar ratio of 350:1.0:0.2 (monomer/CTA/ initator). The polymerization was carried out in a sealed flask at 75 °C and terminated after 6 h by removal of heat and exposure to oxygen. The polymer was then precipitated in diethyl ether and dried under vacuum. The polymerization provided a well-defined (POEGMA-rBP)-b-P4VP diblock copolymer of molecular weight Mn = 29.3 kg mol−1 with a dispersity of 1.11 (by SEC, Figure S5). DP4VP and Mn were determined by averaging the comparison of the 1H NMR (400 MHz, CDCl3) peaks at δ 6.33 (m, 2 H) and 8.26 (m, 2 H) for 4VP and to the 3.31 (s, 3 H) peak for OEGMA (Figure S2). Quaternization of (POEGMA-r-BP)-b-P4VP. The block copolymer was quaternized with a 4-fold molar excess of iodomethane to 4VP monomer in minimal DMF to dissolve the polymer. The reaction mixture was stirred at room temperature for 24 h, and the modified I



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HQ:NSC16/Al BS probe from MikroMasch. Images were processed using the Nova PX software from NT-MDT. Planar or second-order corrections were applied to all images. Film Height Measurement. The film thickness was determined by a scratch test. Films were scratched using a 30 gauge needle (BD). An SPM scan was taken over the scratch and a planar flattening correction was applied to the film surface. The average height profile was then extracted from the data and analyzed to give the average film height. Grazing-Incidence Small-Angle X-ray Scattering (GISAXS). GISAXS experiments were conducted at Argonne National Laboratory at beamline 8-ID-E using X-ray with energy of 10.86 keV. Samples were measured under ambient conditions at three incident angles (αi) 0.14, 0.15, and 0.155° between the critical angle of PEGcoated silicon and the critical angle of the C3M film. The reported images are from an incident angle of 0.155°. Two images obtained at two different detector positions were combined to fill the gaps in the detector. Samples were exposed for 5 s at each angle and detector position, keeping the total exposure time to 30 s. The sample-todetector distance was 2165 mm. Data were analyzed using the GIXSGUI software package written by Dr Zhang Jiang. Data were converted to q space by applying correction parameters (stored as .MAT file format) provided by the beamline scientists. PhoA Activity Assays. PhoA activity was measured fluorimetrically on a TECAN Infinite M200Pro plate reader. 4-Methylumbelliferyl phosphate (4-MUP) (Aldrich) was converted to 4-methumbelliferone (4-MU), which was excited at 362 nm and measured at 448 nm. 4-MUP was dissolved in dimethyl sulfoxide at 10 mM and diluted in water to the concentrations for the assays. Standard curves (Figure S17) for the assays were prepared as described in the SI. Solvatochromic effects due to changes in DMSO concentration were negligible (Figure S18). Assays on the enzyme and micelles were conducted in 96-well plates (Brand, #781602). The mutants were compared in 100 mM 3(N-morpholino)propanesulfonic acid (MOPS) buffer, pH 7.5, supplemented with 500 mM NaCl. The mutants were diluted to 0.5 μg of enzyme L−1 in MOPS buffer. MOPS buffer (160 μL) and 20 μL of substrate solution were added to each well. After taking an initial reading, 20 μL of diluted enzyme was injected into each well. The emission was measured for 2 min. Due to the high salinity of the MOPS buffer, the comparison of free enzyme activity to the micelle activity was conducted in 100 mM Tris at pH 8.0. A standard curve was prepared in this buffer. Micelles were prepared by adding an equivalent mass of POEGMA-b-qP4VP as a protein in solution. The micelles and free enzyme were then diluted to 0.5 μg of enzyme L−1 in Tris buffer. Tris buffer (160 μL) and 20 μL of diluted enzyme or micelles were added to each well. After taking an initial reading, 20 μL of substrate solution was added to each well. The emission was measured for 4 min. Assays on the films were conducted in 12-well plates (Falcon, #353043). The films were secured to the bottom of the wells using a carbon tape; negative controls were conducted to ensure that the tape did not affect the activity (Figure S19). Films were swollen overnight in 20 mM Tris, pH 8.0, with 1 mM MgSO4 and 0.1 mM ZnSO4. Prior to the assay, films were rinsed with water. Tris (900 μL, 100 mM, pH 8.0) was then added to each well. After an initial background measurement, 100 μL of the substrate solution was added. The emission was then measured for 4 min. For metal sensing, the films were first assayed as described above and then exposed to 100 mM EDTA in 100 mM Tris, pH 8.0, for 25− 30 min to convert the enzyme into apoenzyme. Films were rinsed thoroughly with water, and the activity was measured again to ensure inactivation. After another rinse, films were exposed to water containing the specified concentration of analyte and 1 mM MgSO4 for 20−25 min unless otherwise specified. Films were rinsed and assayed to determine the level of reactivation.

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b08478.

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NMR and SEC of polymers, PAGE analysis of proteins, 1D GISAXS traces from films with varied protein loading, GISAXS patterns from films annealed under different solvents, NMR and LRMS of benzophenone monomer, gene and primer sequences, standard curves, and solvatochromic data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (617) 715-4548. ORCID

Allie C. Obermeyer: 0000-0003-2412-2021 Bradley D. Olsen: 0000-0002-7272-7140 Author Contributions

A.C.O. and B.D.O. conceived the immobilization method. A.C.O. and R.J.F. synthesized and characterized materials. H.V.S. conceived and completed further experiments with help from B.D.O., H.V.S., and B.D.O. cowrote the manuscript. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the United States Defense Threat Reduction Agency (HDTRA1-16-1-0038). The gene for PhoA in the pTrc99a plasmid was obtained as a generous gift from Dr Jeff Glasgow.

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REFERENCES

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Catalytic Biosensors from Complex Coacervate Core Micelle (C3M

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