Enzyme Immobilization in Polyelectrolyte Brushes: High Loading and

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Enzyme Immobilization in Polyelectrolyte Brushes: High Loading and Enhanced Activity Compared to Monolayers Gustav Ferrand-Drake del Castillo, Meike König, Martin Müller, KlausJochen Eichhorn, Manfred Stamm, Petra Uhlmann, and Andreas B. Dahlin Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00056 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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Enzyme Immobilization in Polyelectrolyte Brushes: High Loading and Enhanced Activity Compared to Monolayers Gustav Ferrand-Drake del Castillo,1 Meike Koenig,2,† Martin Müller, 2 Klaus-Jochen Eichhorn,2 Manfred Stamm,2 Petra Uhlmann2,3 and Andreas Dahlin.1,*

1 Chalmers University of Technology, Department of Chemistry and Chemical Engineering, 41296 Göteborg, Sweden.

2 Leibniz Institute of Polymer Research Dresden, Hohe Str. 6, D-01069 Dresden, Germany.

3 Department of Chemistry, University of Nebraska-Lincoln, Hamilton Hall, 639 North 12th Street, Lincoln, Nebraska 68588, United States.

KEYWORDS

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polyelectrolyte brushes; enzyme immobilization; surface plasmon resonance; surfaceinitiated polymerization; atom transfer radical polymerization


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ABSTRACT

Catalysis by enzymes on surfaces has many applications. However, strategies for efficient enzyme immobilization with preserved activity are still in need of further development. In this work we investigate polyelectrolyte brushes prepared by both grafting-to and grafting-from with the aim to achieve high catalytic activity. For comparison, self-assembled monolayers that bind enzymes with the same chemical interactions are included. We use the model enzyme glucose oxidase and two kinds of polymers: anionic poly(acrylic acid) and cationic poly(diethylamino)methyl methacrylate. Surface plasmon resonance and spectroscopic ellipsometry are used for accurate quantification of surface coverage. Besides binding more enzymes, the “3D-like” brush environment enhances the specific activity compared to immobilization on self-assembled monolayers. For grafting-from brushes, multilayers of enzymes were spontaneously and irreversibly immobilized without conjugation chemistry. When the pH was between the pI of the enzyme and the pKa of the polymer, binding was considerable (thousands of ng/cm2 or up to 50% of the polymer mass), even at physiological ionic strength. However, binding


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was observed also when the brushes were neutrally charged. For acidic brushes (both grafting-to and grafting-from), the activity was higher for covalent immobilization compared to non-covalent. For grafting-from brushes, a fully preserved specific activity compared to enzymes in the liquid bulk was achieved, both with covalent (acidic brush) and non-covalent (basic brush) immobilization. Catalytic activity of hundreds of pmol cm-2 s-1 were easily obtained for polybasic brushes only tens of nm in dry thickness. This study provides new insights for designing functional interfaces based on enzymatic catalysis.


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INTRODUCTION Enzymes are used in many applications such as industrial food and drug production,1 antimicrobial surfaces,2 drug delivery,3,

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synthetic biological systems,5 biofuel cells,6

biosensors7 and integrated electronic devices.8 Catalysis by help of enzymes is attractive since they operate under mild conditions in aqueous solvents with high selectivity, chiral recognition and compatibility with living systems.1 Recently, there has also been a strong interest in reproducing cascade reactions in confined environments that mimic compartmentalization in cells.9 As with inorganic heterogeneous catalysis, a requirement for practical applications is normally immobilization of the enzymes to an insoluble support that enables easy reuse and continuous collection of products. Upon surface immobilization enzymes may increase their stability and tolerances towards variations in pH, temperature and solvent composition compared to enzymes in the liquid bulk.10 However, most enzymes lose activity upon immobilization,11 which reduces their effectivity in applications. Consequently, enzyme immobilization remains an active area of research11 where the main interest lies in retention of the enzymatic activity in combination with controlling and maximizing the quantity of enzymes on the solid support.


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Polyelectrolyte brushes, i.e. charged macromolecules grafted at high density to a surface,12 can bind high quantities of biomolecules and are thus potentially useful for enzyme immobilization.13,

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The repulsion between the chains energetically favors

stretching away from the surface and the conformations may differ from neutral solvated polymer chains.12 Compared to a self-assembled monolayer (SAM) of small molecules, a polyelectrolyte brush occupies a larger volume and contains a higher number of functional groups. This potentially enables more enzymes to be immobilized per surface area by formation of multilayers.15 Depending on the polymer type, different types of immobilization chemistries can be used, such as attraction due to opposite charges, hydrophobic interactions or the introduction of covalent bonds by conjugation protocols.16, 17, 18, 19

For certain combinations of brushes and proteins, full retention of the secondary

structure of the immobilized enzyme is possible.14, 20 Notably, the brush is also a highly flexible linker in comparison to shorter stiffer organic chains, such as in a SAM. However, further research is needed on the topic of polyelectrolyte brushes for enzyme immobilization. In particular, there is a lack of reports showing the influence from the length of the linker to the surface, while maintaining the same immobilization chemistry.


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A more flexible linker potentially makes the immobilization less invasive with respect to the orientational and conformational freedom of the enzymes, but this effect has not been methodically investigated. Further, there is still a need to test more immobilization strategies for achieving both high enzyme loading (molecules per area) and high specific activity (conversion rate per enzyme). Metal-ion complexation was shown to provide high coverage of ribonuclease A in poly(acrylic acid) (PAA) brushes in comparison with covalent binding, but the activity was reduced compared to enzymes in solution.19 Several other studies have shown immobilization by inherent attractive forces between enzyme and brush, but with the aim of investigating curvature effects,21 developing sensors22, 23 or controlling activity by pH and temperature.14 In such studies the activity has only been compared with enzymes in bulk (if measured at all) and high surface loading is not the primary goal. In this work, we perform a systematic study of how various enzyme immobilization strategies based on polyelectrolyte brushes influence the loading efficiency as well as the specific activity (conversion rate per molecule), using glucose oxidase (GOX) as a model. Importantly, the activity is compared both to that of enzymes in solution and to that of


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enzymes immobilized on SAMs using the same chemical interactions. Thus, we can elucidate the influence from the flexibility and three-dimensionality of the brush environment compared to flat monolayers. Using PAA brushes, the GOX activity is directly compared for covalent vs electrostatic immobilization. In addition, we use for the first time brushes of polybasic poly(diethylamino)methyl methacrylate (PDEA) for spontaneous and highly efficient enzyme immobilization. Functionalization is performed on gold surfaces that are used for surface plasmon resonance (SPR) spectroscopy or silica surfaces that are used for spectroscopic ellipsometry (SE), such that each step can be monitored in real-time. Brushes are prepared by grafting-from (end-grafted) on gold or by grafting-to (side-group grafted) on silica. Dry SPR scans are used for the first time to accurately quantify the surface coverage of polymer and enzyme. The GOX activity is determined by a colorimetric assay in a customized setup. Several complementary techniques are used to verify the results. Our study provides new insights on how the immobilization method influences the specific activity and shows new ways to improve the catalytic activity.


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MATERIALS AND METHODS Chemicals: H2O2 (30%) and NH4OH (28-30%) were from ACROS, while H2SO4 (98%) and ethanol (99.5%) were from SOLVECO. The chemicals for polymerization were the monomers 2-(diethylamino)ethyl methacrylate (inhibitor removed with an alumina column, after which the monomer was stored at -20°C and warmed to room temperature immediately before use), sodium acrylate, solvent methanol, ligand N,N,N’,N’’pentamethyldiethylenetriamine (PMDTA), catalyst CuBr2 and the reducing agent ascorbic acid. The initiator bis[2-(2-bromoisobutyryloxy)undecyl]disulfide (DTBU) was dissolved in ethanol to 2 mM. For preparing thiol SAMs, HS-C11-EG6-OCH2-COOH or HS-C11-EG6OCH2-NH2 was mixed with HS-C11-EG6-OCH2-OH (ProChimia, Poland), in ethanol to 2 mM total and with a composition of HS-R-COOH : HS-R-OH = 2:8 or HS-R-NH2 : HS-ROH = 2:8. PAA Mn = 26 500 g/mol, Mw/Mn = 1.7 was used for grafting-to (Polymer Source, Canada). In enzyme assay preparation and activity experiments, xylenol orange tetrasodium salt, ammonium iron(II) sulfate hexahydrate, β-D-glucose, and GOX Type VII G2133 or G6125 from Asperigillus Niger were used. (Although two different GOX types were investigated, exactly the same GOX was used in all experiments comparing activity.)


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For covalent immobilization of enzymes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and sulfo-N-hydroxysuccinimide (NHS). Buffers used for activity measurements on gold surfaces were phosphate buffered saline (PBS) tablets (0.01 M phosphate, 0.14 M NaCl) or 20 mM 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) with no further salts added. For both buffers the pH was modified to desired values by 1 M HCl or NaOH (change in ionic strength 6° shift), showing that GOX can be efficiently immobilized in the neutral PAA brush, potentially supported by the -COOH groups acting as hydrogen bond donors. Upon raising the pH by switching to PBS buffer most was lost again, which can be expected


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since the enzyme and the polymer obtain the same charge. However, a considerable amount of GOX (~1° signal) does remain bound (Fig. 1C). This is not surprising as it is well-known that proteins may bind to polyelectrolyte brushes even when carrying the same charge due to charged patches on the protein surface41 or local charge regulation.42 At the same time it is interesting to note that we observed full exclusion of weakly positively charged GOX (at pH 4.0) from the positively charged PDEA brush (Fig. 1B). Note that the bulk refractive index change of the buffers is negligible.26 Overall, the data in Fig. 1 shows that for both polyelectrolytes, pH and ionic strength strongly influence the amount of immobilized GOX and thus the ideal strategy depends on the final operating conditions for the enzyme. In this work we optimize GOX immobilization at physiological ionic strength and at pH 6.0 where the specific activity is highest.43 Therefore, the final SPR signals from GOX immobilization after rinsing the chamber with PBS buffer at pH 6.0 are the most relevant. Also, preliminary tests on GOX in neutral brushes showed very low activity, suggesting this is not a viable strategy (example in Supporting Information).


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Figure 1. Monitoring GOX binding. (A) SPR shift (980 nm) as function of time during noncovalent immobilization of GOX at pH 6.0 into PDEA in MES (lower ionic strength) or PBS buffer (physiological ionic strength). (B) SPR shift (785 nm) as function of time during noncovalent immobilization of GOX onto PDEA in PBS buffers with different pH. (C) SPR shift (980 nm) as function of time during non-covalent immobilization of GOX on PAA in MES buffer (pH 4.0). At 110 min PBS (pH 8.0) was injected and at 125 min MES buffer pH 4.0 again. (D) SPR and TIR angle shift (670 nm) as function of time during covalent immobilization of GOX on PAA using EDC/NHS coupling chemistry. The injections indicated by numbers are (I) EDC/NHS in MES (pH 4.0), (II) GOX in MES (pH 4.0) and (III) PBS (pH 8.0). All dashed lines indicate the start and finish of the GOX injection. Note that the SPR wavelength influences the absolute signals and comparisons should only be done within each subfigure.

In order to investigate the role of immobilization chemistry further, GOX was also immobilized in PAA by conventional EDC/NHS conjugation27 (Fig. 1D). Notably, the SPR data reveals less binding of GOX (c.f. Fig. 1C) after the initial activation step, even though


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the buffer used is identical (MES pH 4.0), which is likely related to the esterification of COOH. As expected from efficient covalent linking, all GOX remained after washing with PBS (no signal decrease). The covalent bond formation was further verified by FTIR (Supporting Information), but we could not fully exclude that some of the GOX still was immobilized non-covalently. SPR data provides information about the binding kinetics under different conditions, but exact quantification of the amount of immobilized GOX based on data such as that in Fig. 1 is not straightforward. This is because the concentration profile of immobilized GOX perpendicular to the surface is not known. The SPR sensitivity represented by the evanescent field extension, although somewhat dependent on wavelength,44 is not too different from the brush heights when the polymers are solvated26 (hundreds of nm). To overcome this problem, we performed SPR scans on dry surfaces before and after ATRP as well as before and after GOX binding (Fig. 2). In all cases, the samples were rinsed with PBS at pH 6.0 and also milli-Q before drying. Fortunately, the refractive index of dry proteins is very similar (~1.5) to that of the polymers and thus the films obtained after immobilization were treated as single layers where only the thickness was allowed to vary


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when fitting the spectra to Fresnel models.26 Clearly, the angular reflectivity spectra are excellently reproduced by the calculations (Fig. 2), even with only one parameter free to vary (thickness), which illustrates the high accuracy of the quantification. Importantly, to compare with monolayers, the formation of thiol SAMs and subsequent GOX adsorption was quantified in the same manner. For SAMs we assumed that the GOX layer was on top of the linker due to its high density (we obtained d = 2.0 nm assuming n = 1.45). The presence of polymers and GOX on the gold surfaces was further verified by XPS and FTIR (Supporting Information).


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Figure 2. SPR spectra, i.e. reflectivity as function of incident angle (in degrees) on dried surfaces. (A) PDEA and non-covalent GOX binding. (B) PAA and covalent GOX binding. (C) Alkanethiol-oligo(ethylene glycol) terminated by NH2, non-covalent GOX binding (D) Alkanethiol-oligo(ethylene glycol) terminated by COOH, covalent GOX binding.


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The amount of immobilized GOX for the different grafting-from and SAM functionalization strategies is summarized in Fig. 3. The mass coverage was obtained from the thickness by assuming a density of 1.35 g/cm3 for GOX,45 while 1.22 g/cm3 was used for the polymers.14 We emphasize that the values in Fig. 3 correspond to irreversibly bound GOX at physiological ionic strength (PBS buffer) and at the optimal pH for catalysis (6.0). Also, it is important to be aware of the thickness of the brushes since this influences the amount of immobilized enzymes. For the data in Fig. 3 the dry thickness was 27 nm for PAA and 42 nm for PDEA. Fig. 3A shows that a much higher amount of GOX can be immobilized inside brushes compared to on SAMs and the coverage clearly corresponds to multilayers of GOX (molecular weight 160 kg/mol). For instance, the PDEA brush with dry thickness 42 nm has at least 9 GOX molecules bound on a 10 × 10 nm2 area. It seems likely that the enzymes are entangled throughout by the polymer chains also in the dry state rather than forming a separate film, but since the refractive index values are so similar it has no relevance on the analysis. The quantification offered by the dry state SPR spectra thus confirms the “3D nature” of the immobilization strategies based on graftingfrom brushes. In contrast, the coverage on SAM-modified gold agrees well with a GOX


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monolayer with reasonably high packing density (~90 nm2 per molecule). Note that a critical parameter which likely influences the GOX binding is the grafting density of the brushes (number of coils per area). This cannot easily be determined, at least not in grafting-from methods.46 However, we can quantify both mass coverage and thickness.26 Further, due to the similar polymerization rate of the ATRP brushes26 we expect the grafting density to be quite similar (although unknown) for PAA and PDEA. We also normalized the GOX coverage to the mass coverage of the initial surface coating, i.e. polymer or SAM (Fig. 3B). Using this dimensionless quantification, the comparison between brushes becomes fair even if they differ in thickness. (The SAMs appear efficient for immobilization simply because they are only 2 nm in thickness.) PDEA is clearly more efficient for immobilizing GOX, regardless of whether the bonds to PAA are covalent or not. Furthermore, the covalent immobilization in PAA gives almost twice as much GOX on the surface compared to the non-covalent method. Both covalent (COOH activation) and non-covalent (-NH3+ electrostatic) immobilization provides approximately the same amount of GOX on SAMs. Here the established alkanethiol SAM density of 0.84 g/cm3 was used to quantify mass coverage.47


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Figure 3. Comparison of enzyme coverage determined by SPR for immobilization strategies based on gold surfaces. (At least 3 unique surfaces were analyzed for the SAMs and 5 for brushes.) The dry PAA thickness was 27±2 nm and the dry PDEA thickness was 43±4 nm. (A) Results in absolute values (ng/cm2). (B) Coverage when


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normalized to the mass coverage of the brush or the SAM (dimensionless). Error bars represent ± one standard deviation.

In order to investigate the effects from brush morphology we also used sidegroupgrafted PAA (Guiselin) brushes prepared by grafting 26.5 kg/mol PAA to polymermodified silica,14 resulting in a coverage of 610 ng/cm2. These brushes differ considerably in internal structure as it is the side-groups which are covalently linked to the surface,24 which leads to a lower conformational entropy for the chains compared to end-grafting of the same number of molecules. The Guiselin brushes are also thinner (6 nm dry thickness) than the ATRP brushes, but swell considerably upon hydration and deprotonation.48 The immobilization of GOX to Guiselin PAA brushes was monitored using in-situ SE measurements and quantified after each immobilization step (Supporting Information). A solution pH of 6.0 was used for EDC/NHS activation and GOX binding since we have previously demonstrated that this leads to very low non-covalent immobilization for GOX in these brushes.14 The quantity of GOX covalently coupled to the Guiselin PAA brush prepared by grafting-to was determined to be ~300 ng/cm2 by a


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modified de Feijter approach.49, 50 Thus, when normalized to the polymer coverage, the immobilized GOX amount is similar to the best result obtained with grafting-from (~0.5 g GOX per g PDEA, Fig 3B). The absolute amount is comparable to the coverage of GOX on SAMs with -COOH terminated functional groups and the same covalent immobilization (314±59 ng/cm2, Fig. 3A). After quantifying the bound amount of GOX on the different brushes, we investigated how the specific activity of the enzymes on the surface, i.e. the conversion rate per molecule, depends on the immobilization method. A colorimetric activity assay based on quantification of the amount of produced H2O2 was developed as illustrated in Fig. 4.14 In this study we did not include directly physisorbed enzymes since such immobilization is well-known to lead to relatively poor activity,39 but we did compare with the activity of free enzymes in solution. The amount of GOX in the droplet for the bulk activity measurements was determined by UV absorbance and the extinction coefficient specified by the manufacturer (2.67×105 M-1cm-1). This gave concentrations that correspond to a GOX content of 78% by weight in the purchased product (specification for Type VII G2133


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>60%). The catalytic reaction (Scheme 3) can be regarded as irreversible since Dgluconolactone rapidly decomposes by hydrolysis.36 The functionalized surfaces were possible to store and reuse, i.e. rinsing and applying a new droplet containing glucose resulted in the same catalytic activity. Still, we observed reduced catalytic activity after longer times during an assay (Fig. 4D). This effect must therefore be associated with the reactants or products, not the enzymes. Since our method uses a static droplet where the reaction medium volume is comparably low, oxygen depletion within the droplet can be expected.52 After 15 min reaction, based on the amount of H2O2 produced, we estimated that typically ~20% of the oxygen initially present in the droplet (equilibrium with air at room temperature) is consumed by GOX, while as much as 98% of the glucose added remains present in the reaction medium. It is possible that a concentration gradient of oxygen is established within the droplet for the surface confined catalysis, which reduces the conversion rate with time. Alternatively, the reduced activity with time may be attributed to a high local concentration of H2O253 (selfpoisoning) at the surface. Regardless, this does not influence our comparisons of


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immobilization methods and implies that the activity, especially for immobilized GOX, may be even higher for a steady-flow system that supplies glucose and oxygen.

Figure 4. Description of GOX activity measurements with grafting-from brushes. See also Scheme 3. (A) A small volume is sampled from the droplet in contact with the surface. (B) Assay absorption spectra for different concentrations of H2O2. (C) Absorbance calibration


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curve for H2O2 concentration. (D) Example data of concentration of H2O2 in droplet increasing over time. The initial activity is defined from a linear fit to the first 5 min.

The activity measurements are summarized in Fig. 5 as initial reaction rate (linear fit to the first 5 min) as well as amount of H2O2 produced after 15 min, in both cases normalized to the amount of enzyme on the surface area exposed to the droplet in the well (Fig. 4A). The activity is generally in good agreement with previous studies of GOX.36,

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For

instance, at the same glucose concentration as in this study, initial rates of 17 s-1 have been obtained,54 similar to our value of 19 s-1. Clearly, the brush-based immobilization strategies are superior not just for high loading, but they also preserve enzyme activity much more than SAM monolayers with the same immobilization chemistry. For PDEA as well as covalent binding to PAA, the activity is the same as the free enzyme in solution, or the difference is very small (Fig. 5). In comparison with literature, retention of activity of covalently immobilized GOX of 60% relative to the bulk solution value was reported for poly(glycidyl methacrylate) and for poly(2-vinyl-4,4-dimethyl azlactone) brushes.16, 17 In polypyrrole films the activity has been reported to be 36% of the bulk value.55 There are


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some examples in the literature where enzymatic activity is highly preserved13 or even enhanced56 upon immobilization in polyelectrolytes. Similarly, attaching zwitterionic chains have been used to stabilize and enhance enzymes in solution.57 We consider the fully preserved activity of GOX upon immobilization observed here as a good complement to previous studies since it has been measured on a planar surface with highly accurate dry state SPR quantification. GOX immobilized on SAMs has at best been reported to retain 50% activity relative to bulk, which is a considerable reduction and still higher than what we observe.58 Note that one possible source of error in our analysis is that O2 depletion should be more pronounced when the absolute amount of GOX is higher. This error would mean that the specific activity comparison favors GOX on SAMs as they have much lower absolute coverage (Fig. 3A). The comparison between brushes and bulk solution measurements is still valid since the total amount of GOX was chosen to be approximately the same as in the brushes and a gradient in O2 concentration will result in lower activity in the brushes.


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Figure 5. Comparison of GOX activity for the different immobilization strategies and in bulk. Activity measurements were performed in PBS at pH 6.0 with [glucose] = 0.01 M and 25 Cº. Each measurement was repeated at least in triplicate. (A) Activity presented as initial rate (conversions per second) during the first 5 min. (B) Activity as amount of H2O2 per amount of GOX after 15 min. Error bars represent ± one standard deviation.


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Results of Michaelis-Menten analysis are shown in Fig. 6. The values of KM are an order of magnitude lower than what is expected when glucose binding is limiting the reaction and an order of magnitude higher than for oxygen limitation.54 This is in agreement with limited O2 content in the droplet influencing (but not limiting) the reaction rate. If the droplet on the surface where the enzymes are immobilized is treated as a homogenous system, the effective enzyme concentration is typically ~50 nM (depending on brush). Due to this very low concentration the Briggs-Haldane constant is more applicable for our system (Supporting Information). The values for kcat, i.e. the rate constant of the catalytic conversion step, are somewhat reduced compared to optimal GOX operation. It has been known for a long time that the enzyme can perform hundreds of conversions per second under ideal conditions.54 The discrepancy can again be attributed to limited O2 supply in the droplet. (Even our “initial” rate is based on a relatively long time of 5 min.) In any case, for the purpose of this study it is more important to note that the values of KM and kcat obtained in solution do not differ much from those obtained for GOX in polyeletrolyte brushes (Fig. 6). This confirms that the enzyme behavior is not significantly altered inside the brush.


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Figure 6. Michaelis-Menten plots of product formation rate (M/s) vs glucose concentration for GOX in PAA, GOX in PDEA and bulk GOX. The fitted parameters KM and kcat are shown for each case. Error bars represent ± one standard deviation.

Results from activity measurements on PAA Guiselin brushes are shown in Fig. 7. In these experiments another batch of GOX was used and the assay was performed slightly differently,14 so the absolute activity is not to be compared with Fig. 5. Instead, we point out that the GOX activity in these PAA brushes is quite reduced compared to solution, despite the fact that the immobilization chemistry is identical to that in brushes prepared via grafting-from. This suggests that the brush morphology also plays an important role


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for the specific activity. The brush morphology may influence the structure and structural freedom of the immobilized GOX, e.g. through the number of interaction points. An alternative explanation which we consider less likely is that it affects reactant transport during the catalysis, but the volume fraction of polymer inside the Guiselin brushes is similar to the grafting-from brushes. Furthermore, from Fig. 7 it is possible to confirm the effect on activity due to covalent vs non-covalent immobilization to PAA, i.e. a factor of ~2 is observed just as for the brushes prepared by ATRP (Fig. 5). It is arguably a bit counterintuitive that covalent bonds would be less invasive for the enzyme as the secondary and tertiary structure should be less perturbed by weaker interactions. Altered structure is indeed generally linked to activity loss,14, 58 but exceptions do exist, especially for GOX.40, 58 Also, covalently grafted zwitterionic chains may enhance activity of enzymes in solution.57 Regardless, our results show that even if covalent bonds are preferable to promote activity in PAA, the ideal strategy for high activity per area is actually non-covalent immobilization in PDEA since it binds the highest amount (GOX mass is 50% of brush mass) in addition to preserving the activity. Spontaneous immobilization without conjugation chemistry is naturally also


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easier to perform and works well at pH 6-7 (positively charged PDEA brush) as many enzymes have a pI slightly lower than that (following the bimodal pI distribution). We did measure spontaneous and irreversible immobilization in PDEA brushes at pH 6.0 also for several other enzymes such as horseradish peroxidase (2687±477 ng/cm2) and βgalactosidase (2366±285 ng/cm2). It was even possible to immobilize different enzymes at once and then monitor cascade reactions (results to be published separately).


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Figure 7. Comparison of the activity of GOX in solution after, covalently coupled to and non-covalently adsorbed to PAA brushes prepared by sidegroup grafting-to. The amount of H2O2 produced after 15 min was analyzed. The surface coverage of PAA was 610 ng/cm2.

Although optimizing enzymatic activity is interesting from an application point of view, we also wish to investigate the fundamental question why brushes provide better specific activity compared to SAMs or directly adsorbed enzymes. The preserved activity shows that the hydrated brushes in their charged state at pH 6.0 clearly do not limit reactant transport to a significant extent, as expected since the volume fraction of solvent is high.26 The 3D brush environment, in contrast to a SAM, naturally provides interactions points everywhere on the enzyme surface and intuitively this should be more invasive. Still we observe much higher specific activity in the brush environment, suggesting that the length of the tethering molecules between the enzymes and the surface plays a more important role. The contour lengths of the ATRP chains are on the order of hundreds of nm, the SAM thiols (with 6 ethylene glycol units) merely a few nm and the Guiselin brushes lie


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somewhere in between. A longer link is more flexible and allows for more conformations which should increase the probability of initiating the catalytic conversion. In contrast, enzymes on top on a planar SAM (or physisorbed) lack rotational freedom entirely.11 CONCLUSIONS In summary we investigated different immobilization strategies for GOX based on polyelectrolyte brushes, with the aim to elucidate the role of the (3D) brush environment compared to monolayers (2D). SPR was used as the main tool for investigating the immobilization in real-time and was shown to provide accurate quantification by angular spectra obtained in the dry state. In addition, we have tried to maximize both enzyme loading and catalytic activity. Brushes are superior to SAMs in all respects, as we could verify by using the same immobilization chemistry. For PAA, end-grafted brushes prepared by ATRP are preferable over sidegroup-grafted Guiselin brushes, although the latter do not require surface-initiated polymerization. The enzyme binding capacity is strongly dependent on ionic strength and pH, but electrostatic attractions are not solely responsible for the non-covalent immobilization. For grafting-from brushes the specific activity of GOX can be fully preserved, either by covalent binding to the acidic brush or


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by non-covalent immobilization in the basic PDEA brush. For highest possible GOX activity per surface area at physiological conditions, PDEA is an excellent polymer and offers spontaneous immobilization without conjugation chemistry. Preliminary results indicate that PDEA is suitable for immobilizing many other enzymes as well. One major strength of this study is that the same chemical interactions have been used to bind the enzyme in the brushes as on the monolayers. Remarkably, even though the surrounding brush leads to interactions with the whole enzyme surface it is apparently still less invasive than having the same chemical interaction occurring at fewer contact points in one plane. Speculatively, the hydrated brushes are more similar to native cell environments for enzymes as compared to dense SAM surfaces. The results also suggest that a longer and thus more flexible linker between enzyme and surface is highly important for preserving activity. However, further work is needed to confirm this for more polymers and other enzymes. In future work the surface functionalization protocols developed here may be extended to multiple enzymes for cascade reactions and more extensive synthesis schemes.


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ASSOCIATED CONTENT Supporting Information GOX quantification in bulk solution, further activity measurements data, IR spectra, XPS spectra, SE measurements, details on Michaelis-Menten.

AUTHOR INFORMATION * Corresponding author: [email protected] † Present address: Karlsruhe Institute of Technology, Institute of Functional Interfaces, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany. Author Contributions All authors have contributed to the results and the writing of the manuscript. Approval of this submission has been given by all coauthors. Funding Sources Financial support was granted by the Knut & Alice Wallenberg Foundation (Academy Fellow 2015.0161), the Swedish Research Council (project grant 2016-03319), the German Science Foundation (DFG) within the DFG-NSF cooperation project (DFG Proj.


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Nr. STA 324/49-1 and EI 317/6-1) in the frame of the “Materials World Network” and in the frame of the priority program SPP 1369 “Polymer-Solid Contacts: Interfaces and Interphases” (DFG Proj. Nr. STA 324/37-1).

ACKNOWLEDGMENT We thank Dr Raphael Zahn at the Swiss Federal Institute of Technology Zürich for unraveling (at least some of) the mysteries of polyelectrolytes interacting with proteins.

ABBREVIATIONS SPR, surface plasmon resonance; GOX, glucose oxidase; PAA, poly(acrylic acid); PDEA,

poly(diethylamino)methyl

methacrylate;

EDC,

1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide, SAM; self-assembled monolayer, ARGET ATRP; activator regenerating electron transfer atom transfer radical polymerization.


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Enzyme Immobilization in Polyelectrolyte Brushes: High Loading and

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