Stable and Simple Immobilization of Proteinase K Inside Glass Tubes

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Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels Andreas Küchler,† Julian N. Bleich,† Bernhard Sebastian,‡ Petra S. Dittrich,‡ and Peter Walde*,† †

Polymer Chemistry Group, Department of Materials (D-MATL), ETH Zürich, Vladimir-Prelog-Weg 5, 8093 Zürich, Switzerland Bioanalytics Group, Department of Biosystems Science and Engineering (D-BSSE), ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland

‡

S Supporting Information *

ABSTRACT: Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acids from biological samples, or in proteomics and prion research. Toward applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer− enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on nonspecific, noncovalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 °C and during continuous operation at 25 °C and pH = 7.0. The procedure developed was successfully applied for the immobilization of proK on a glass/PDMS (polydimethylsiloxane) microchip, which is a requirement for applications in the field of proK-based protein analysis with such type of microfluidic devices. KEYWORDS: enzyme, protease, immobilization, adsorption, dendronized polymer, conjugate, flow reactor, microchip enzymes are first conjugated to the dendronized polymer (denpol) de-PG2 (Figure 1), followed by simple adsorption of the denpol−enzyme conjugate from an aqueous solution.17,18 In both of these previous cases with HRP or GOD, the enzymes were linked to the denpol via stable bis-aryl hydrazone (BAH) bonds.19,20 With this novel enzyme immobilization approach, a several hundred nanometer long macromolecular object, the denpol−enzyme conjugate, could be deposited on glass surfaces without any detectable desorption because of the many unspecific noncovalent interactions between the large conjugate and the surface. In the study presented here, we investigated whether the concept elaborated previously for the immobilization of the two glycosylated oxidative enzymes HRP and GOD17,18 can also be applied for the immobilization of a proteolytic enzyme, Engyodontium album proteinase K, formerly known as Tritirachium album proteinase K,21 abbreviated as proK. ProK does not contain glycosidic chains. As in the case of HRP and GOD, proK was chosen as model enzyme. ProK is commercially available in pure form, and it is widely applied as efficient catalyst for the degradation of protein/enzyme impurities, for example, during the preparation of nucleic acids

1. INTRODUCTION There are many established procedures for the immobilization of enzymes on solid surfaces.1−13 Often, a solid support is first modified with spacer molecules to which the enzymes are covalently bonded. Alternatively, a solid support is first coated with an organic layer of amphiphiles, followed by the attachment of the enzymes to this layer. The enzyme attachment to the layer may occur through covalent bonds or through very specific noncovalent bonding with the biotin− avidin (or streptavidin) system.14 In this latter case, avidin (or streptavidin) serves as macromolecular linker unit. For glycosylated enzymes, concanavalin A can also be used as another type of specific noncovalent macromolecular linker.15 Most importantly, if the organic layer remains stably adsorbed on the solid support, the enzymes are expected to remain on the surface as well. In contrast to covalent or specific noncovalent macromolecular bonding, the unspecific noncovalent adsorption of enzymes,1,2,4,16 either directly on a solid support or on a previously formed organic coat, usually does not lead to very stable enzyme immobilization; the enzyme molecules tend to desorb from the surface upon storage or during operation. However, we have recently shown in the case of horseradish peroxidase isoenzyme C (HRP) and Aspergillus sp. glucose oxidase (GOD) that a stable unspecific noncovalent immobilization of enzymes on SiO2 surfaces is still possible if the © 2015 American Chemical Society

Received: October 1, 2015 Accepted: November 4, 2015 Published: November 4, 2015 25970

DOI: 10.1021/acsami.5b09301 ACS Appl. Mater. Interfaces 2015, 7, 25970−25980

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ACS Applied Materials & Interfaces

studies on the immobilization of proK for these and other types of applications.29−35 The focus of our work was first on the preparation of a dePG2-proK conjugate in which the proK molecules are bound along the denpol chain via BAH bonds, abbreviated as de-PG2BAH-proK, and then on the immobilization of the prepared dePG2-BAH-proK conjugate on silicate glass microscopy coverslips and inside glass micropipettes (Figure 1) for their continuous use as flow reactors. On the basis of the simplicity of the immobilization methodology and based on the revealed high operational stability of proK immobilized inside glass micropipettes, as found during the course of our work, preliminary successful tests were also made on the immobilization of the conjugate inside the channels of a microfluidic chip. The denpol used in this work had an average length of about 2000 repeating units (r.u.), with a number-average molar mass (basic form, i.e., not including counterions) of ≈1.6·106 g/mol, and a PDI of ≈ 4.3.36 This deprotected denpol is abbreviated as de-PG22000 (Figure 1).

2. EXPERIMENTAL SECTION 2.1. Materials. Engyodontium album proteinase K (proK, recombinant from Pichia pastoris, EC 3.4.21.64, catalog number 03115879001, lot 14321500, and lot 1016630) was obtained from Roche Applied Science (Switzerland). de-PG2 (Pn ≈ 2000, PDI ≈ 4.3), N-succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic) and N-succinimidyl 4-formylbenzoate (S-4FB) were synthesized as described earlier.36,37 Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanylpara-nitroanilide (Suc-AAPF-pNA) and succinyl-L-alanyl-L-alanyl-Lalanyl-L-p-nitroanilide (Suc-AAA-pNA) were purchased from Bachem (Switzerland). BODIPY-FL-casein was obtained from Life Technologies (Switzerland). 2(N-Morpholino)ethanesulfonic acid (MES) and 3(N-morpholino)propanesulfonic acid (MOPS) were obtained from AppliChem (Germany). N,N-dimethylformamide (extra dry over molecular sieve) was obtained from Acros Organics (Switzerland). Amicon ultrafiltration devices were obtained from Merck Millipore or Sigma-Aldrich. Centrisart I ultrafiltration devices were from Sartorius (Switzerland). Round microscopy glass coverlips (diameter 8 mm) were obtained from Science Services (Germany), and glass micropipettes (intraMARK, 200 μL; length = 140 mm; inner diameter = 1.6 mm) were from Brand (Germany). 2.2. Analytical Methods. High resolution UV/vis spectrophotometric measurements were performed using a Jasco V670 spectrophotometer equipped with a water cooled Peltier cuvette holder and quartz cuvettes from Hellma Analytics: 110-QS (1 mm path length,

Figure 1. Schematic representation of the immobilized denpol− enzyme conjugate de-PG2-BAH-proK, indicating the chemical structure of the deprotected second generation denpol de-PG2, the bis-aryl hydrazone linker moiety BAH, and proK. The average number of repeating units (r.u.) of the denpol used was n = 2000. Please note that not all de-PG2 repeating units of the conjugate carried a proK molecule. ProK: PDB code 1IC6.

from biological samples where nucleic acids-degrading enzymes (e.g., DNases) need to be eliminated22 or in the field of proteomics23−27 and prion protein research.28,29 Somewhat surprisingly, however, there are not (yet) many published

Figure 2. Modification of proK with the 4FB linker. The activated S-4FB linker was reacted with nucleophiles present on the surface of proK, presumably mainly amino groups of lysine side chains or the N-terminal amine. A large excess of S-4FB had to be used in order to obtain a reasonable modification ratio of approximately 1 linker/proK. The requirement for a large amount of S-4FB is possibly due to a hydrolytic activity of the active site of proK toward the linker reagent. 25971


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ACS Applied Materials & Interfaces 350 μL), 114-QS (10 mm path length, 1.4 mL), and 105.202-QS (10 mm path length, 50 μL). Concentration determinations were carried out by using a NanoDrop ND-1000 spectrophotometer, and enzymatic activity measurements were performed on a Analytik Jena Specord S600 spectrophotometer using disposable polystyrene cuvettes from Brand (Germany). Online UV/vis spectrophotometric measurements in continuous flow systems were performed using a USB2000+ spectrophotometer (OCOUSB2000+VIS-NIR, 350−1100 nm), a tungsten halogen light source (OCOHL-2000-HP-FHSA) and a Z-flow cell (OCOFIA-Z-SMA-ML-TE) connected with 400 μM optical fibers (OCOQP400-2-UV-BX), all from Ocean Optics, obtained via GMP SA (Switzerland). The de-PG2-BAH-proK adsorption measurements were carried out with a transmission interferometric adsorption sensor (TInAS) developed by Heuberger and Balmer.38,39 The ESI-MS analysis was performed by the MS Service of the Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich. 2.3. Preparation of de-PG2-HyNic. de-PG2-HyNic was prepared as described previously,17,36,37 using a de-PG2 polymer with a numberaverage length of 2000 r.u., de-PG22000 (Figure S1). For the preparation of de-PG22000-HyNic, the same procedure was used as described by Küchler et al.17 2.4. Preparation of ProK-4FB. ProK was dissolved at a concentration of 10 mg/mL in 10 mM MOPS buffer (pH = 8.0) and the exact concentration determined UV/vis spectrophotometrically (ε280 nm (proK) = 41 000 M−1 cm−1).40 The modification reaction was performed in 10 mM MOPS buffer (pH = 8.0) at a proK concentration of 50 μM, using 120 equiv of S-4FB from a 100 mM stock solution in dry DMF (Figure 2). After 4 h at room temperature, the hydrolyzed linker reagent was removed by repetitive ultrafiltration with 10 mM MES buffer (pH = 4.7) using an Amicon Ultra-4, 10 kDa NMWL ultrafiltration device. The obtained proK-4FB solution was stored at 4 °C until further use. For the mass spectrometric characterization of proK-4FB, the samples were desalted by repetitive ultrafiltration with Milli-Q water using an Amicon Ultra-0.5, 10 kDa NMWL ultrafiltration device. UV/vis spectrophotometric quantification of the 4FB linker bound to proK was performed as described by Solulink,41,42 with slight modifications. Derivatization of the 4FB linker moiety with 500 μM 2hydrazinopyridine was carried out in 10 mM MES buffer at pH = 4.7 for 3 h at room temperature. UV/vis spectra were recorded immediately after addition of proK-4FB and after 3 h, and the formation of the BAH bond determined from the increasing absorbance at 350 nm (ε350 nm = 24 500 M−1 cm−1).41 The proK concentration in the proK-4FB solution was quantified with the Bradford protein assay (Sigma-Aldrich, Switzerland),43 as described by the supplier. A calibration was made with known amounts of native proK. 2.5. Preparation of de-PG2-BAH-proK. de-PG2-HyNic and proK-4FB were added to MES buffer (10 mM, pH = 4.7) to a final concentration of 100 μM HyNic and 50 μM 4FB (Figure S2) in a 1 mm path length quartz cuvette. The cuvette was mounted in a Peltier controlled cuvette holder (T set at 25 °C) and the formation of dePG2-BAH-proK was monitored in situ by UV/vis spectrophotometry, recording spectra at intervals of 5 min for 6 h. After recovery of the reaction mixture from the cuvette, free proK and proK-4FB were removed by repetitive ultrafiltration with MOPS buffer (10 mM, pH = 7.0) using a Centrisart I, 300 kDa MWCO ultrafiltration device. The purified de-PG2-BAH-proK was stored in MOPS buffer solution at 4 °C until further use. 2.6. Immobilization of de-PG2-BAH-proK on Silicate Surfaces. 2.6.1. Transmission Interferometric Adsorption Sensor (TInAS) Measurements. TInAS sensors were cleaned and mounted as described before.17 All measurements were carried out at a flow rate of 20 μL/min. Whenever a solution was exchanged with another one, the flow rate was increased temporarily to 400 μL/min for 45−60 s to minimize mixing of the solutions in the flow cell. Afterward, the flow rate was readjusted again to 20 μL/min. After a baseline measurement using MOPS buffer (10 mM, pH = 7.0), a solution of de-PG2-BAHproK (1 μM proK concentration as determined from the BAH

absorption band) in MOPS buffer (10 mM, pH = 7.0) was injected into the TInAS flow cell and the adsorption on the SiO2 surface of the TInAS sensor was monitored. When no further conjugate adsorption on the sensor surface was observed, the MOPS buffer was injected into the flow cell and the level of the adsorbed mass was further monitored to probe possible desorption of the adsorbed material. The evaluation of the raw adsorption data was performed as described by Sannomiya et al.39 The value for the refractive index increment, dn/dc (conjugate) = 0.173 cm3/g, was calculated as linear combination of the contributions of the denpol (29 mass %, dn/dc (denpol) = 0.140 cm3/g)44 and proK (71 mass %, dn/dc (proK) = 0.186 cm3/g).45 2.6.2. Immobilization on Microscopy Glass Coverslips. Round microscopy glass coverslips with a diameter of 8 mm and a total surface of 1 cm2 (both sides) were cleaned by bath sonication in ethanol (3 times 10 min) and dried with nitrogen gas. The cleaned coverslips were transferred into 2 mL polypropylene reaction tubes and the surface wetted with 10 mM MOPS buffer (pH = 7.0). For the adsorption of the de-PG2-BAH-proK conjugate, the buffer solution was removed from the reaction tubes containing the glass coverslips, and a solution of de-PG2-BAH-proK was added (1 μM proK concentration as determined from the BAH absorption band in 10 mM MOPS buffer, pH = 7.0). After 1 h, the de-PG2-BAH-proK solution was removed from the reaction tubes and the coverslips in the reaction tubes were washed 3 times with the MOPS buffer. The dePG2-BAH-proK coated glass coverslips were stored immersed in buffer (10 mM MOPS, pH = 7.0) in the reaction tubes at 4 °C until further use. 2.6.3. Immobilization Inside Glass Micropipettes. Glass micropipettes were cleaned by bath sonication in ethanol (3 times 10 min) and dried with a flow of nitrogen gas. The inner surface was wetted by aspiration of MOPS buffer (10 mM, pH = 7.0). After the buffer was dispensed from the micropipette, the de-PG2-BAH-proK conjugate was adsorbed on the inner surface of the micropipette by aspiration of a de-PG2-BAH-proK solution (1 μM proK concentration as determined from the BAH absorption band, in 10 mM MOPS buffer, pH = 7.0). After incubation for 1 h, the conjugate solution was dispensed from the micropipette and residual conjugate, which did not adsorb, was removed by washing 3 times with 10 mM MOPS buffer (pH = 7.0). Finally, the micropipettes with adsorbed de-PG2-BAHproK were filled with MOPS buffer solution and stored at 4 °C until further use. 2.7. ProK Activity Measurements. 2.7.1. Dissolved ProK. Activity measurements of dissolved proK, proK-4FB and de-PG2BAH-proK were performed in 10 mM MOPS buffer, pH = 7.0, using Suc-AAPF-pNA as chromogenic substrate (Figure S3a). From a stock solution of 50 mM Suc-AAPF-pNA in DMF, 10 μL were added to 990 μL of a proK solution in a disposable polystyrene cuvette. UV/vis spectra of this assay mixture were recorded in intervals of 15 s for 5 min, and the initial velocity was obtained from the rate of the increasing absorbance of p-nitroaniline at λ = 410 nm. A calibration curve correlating the hydrolytic activity to the enzyme concentration was obtained with known amounts of dissolved native proK in a range of 0.1−2.0 nM (Figure S3b). The determination of kcat and KM was performed by using solutions of 5 μM proK and proK-4FB and 16.5 μM de-PG2-BAH-proK (the given concentration is the concentration of proK in the conjugate, as determined through a quantification of the BAH concentration). Suc-AAPF-pNA concentrations of 30−250 μM were used and additional DMF was added for maintaining a constant final concentration of 1 vol % DMF in the assay solution. UV/vis spectra were recorded at intervals of 5 s for 1 min and the initial velocities obtained were quantified by taking into account the UV/vis absorption of p-nitroaniline (ε410 nm = 8800 M−1 cm−1).46 2.7.2. Immobilized de-PG2-BAH-proK. The quantification of the activity of the immobilized proK was performed by addition of 1 mL of substrate solution into 2 mL polypropylene reaction tubes containing a microscopy coverslip with 1 cm2 surface with adsorbed de-PG2-BAHproK. A substrate solution of 500 μM Suc-AAPF-pNA in 10 mM MOPS buffer, pH = 7.0, including 1 vol % DMF, was used. Immediately before adding the substrate solution, the pH = 7.0 storage buffer (10 mM MOPS) was removed from the polypropylene reaction 25972


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Figure 3. ESI-MS analysis of native proK (a and b) and of 4FB-modified proK, as obtained using a molar proK/S-4FB ratio of 1/120 (c and d). Spectra of the multiply charged species (a and c) and of the deconvoluted spectra (b and d) clearly identified the modified proK-4FB, with only a very small intensity corresponding to the proK-(4FB)2. For panels c and d: remaining unmodified, native proK was not removed during the purification and was still present in the sample. For panels b and d: the measured values are indicated together with the calculated values (given in parentheses). tubes. The reaction tubes containing the coverslips and the substrate solution were inverted gently every minute, and after 5 min, the substrate solution was removed from the reaction tubes and the coverslips were washed with buffer (10 mM MOPS, pH = 7.0) and stored at 4 °C until further use. The p-nitroaniline concentration in the assay solution was determined by measuring the absorption at λ = 410 nm. Correlation of the product concentration measured in the assay solution with the rate of product formation measured for different amounts of native proK in solution under the same conditions (see section 2.7.1) allowed the determination of an apparent concentration of active proK on the glass surface, Γapp. For the determination of the operational stability of the immobilized proK, silicate glass micropipettes with adsorbed de-PG2-BAH-proK were installed in a setup with a Z-flow cell for UV/vis spectrophotometric online detection after the outlet of the micropipette. A peristaltic pump (Pharmacia LKB pump P-1) was connected after the Z-flow cell for setting the flow rate. The operational stability of the immobilized proK was studied at a continuous flow rate of 35 μL/min using a substrate solution of 100 μM Suc-AAPF-pNA in MOPS buffer, pH = 7.0. Product formation was detected as steady state level of p-nitroaniline at λ = 410 nm. 2.8. On-Chip Immobilization of de-PG2-BAH-proK. 2.8.1. Microfluidic Chip Preparation. The design of the chip consisted of a single microchannel of 30 cm length, 100 μm width, and 40 μm height with a front inlet, a side inlet and an outlet (see 3.7). The channel runs in a meandering fashion consisting of 14 turns (with 1.84 cm run length between each turn) at a total length of 30 cm, which ends in a single outlet channel about 2 cm downstream of the last turn. The side inlet was connected to the microchannel immediately after the sixth turn. Thus, fluids entering the channel through the front inlet run through its entire length, whereas fluids entering the channel through the side inlet run through roughly the second half of the chip only. The microchannel was produced by cast-molding from a patterned silicon wafer using polydimethylsiloxane (PDMS, Sylgard 184 Dow, Midland, Michigan, USA). Prepolymer and curing agent were mixed 10:1 (w/w), degassed, and poured on the patterned silicon wafer, degassed again, and finally baked at 80 °C for 1 h. The silicon wafer was produced in a clean room by standard soft lithography processing. The PDMS chip was detached from the silicon wafer, cut, and connection holes were punched with a 1.5 mm diameter biopsy

puncher. The chip was cleaned with soapy water (prepared from household detergent) and ethanol, rinsed with ultrapure water and dried. A coverslip was cleaned with soapy water, acetone, 2-propanol and ethanol, rinsed with ultrapure water, and dried. The PDMS chip and the coverslip were loaded in a plasma cleaner (Harrick Plasma, PDC-32G) for surface-activation in an air plasma for 45 s. The activated surfaces were brought into contact for assembly, and the assembled chip was put on a 50 °C hot plate for 12 min for bonding. 2.8.2. Immobilization of de-PG2-BAH-proK in the Microchannels of the Chip. The chip was primed with MOPS buffer (10 mM, pH = 7.0) by centrifugation (Sigma 3-18K) at 800g for 5 min. A 250 μL syringe (Agilent, Santa Clara, California, USA) was filled with a solution of de-PG2-BAH-proK (2 μM proK, in MOPS buffer, pH = 7.0). Another syringe (500 μL, Agilent, Santa Clara, California, USA) was filled with 400 μL of a BODIPY-FL-casein solution (20 μg/mL, in MOPS buffer, pH = 7.0) and a plug of 100 μL MOPS buffer at the tip of the syringe. The syringes were connected to the microfluidic chip with PTFE tubing and the flow was controlled with a syringe pump (Nemesys, Cetoni, Korbussen, Germany). The microfluidic chip was mounted on a widefield inverted microscope (Olympus IX-70) equipped with a 4× objective (Olympus Plan, NA 0.1) that is set up for bright light and fluorescence imaging. For fluorescence imaging, a metal halide lamp in combination with a 470 nm/40 nm excitation filter, a 495 nm dichroic mirror, and a 525 nm/50 nm emission filter were used. Image acquisition was done using an Andor iXon EMCCD (DU 887 BV) camera. First, the conjugate was pumped through the microfluidic channel downstream of the side inlet at a flow rate of 2 μL/min for 60 min and then incubated at stopped flow for 30 min for allowing the conjugate to adsorb (see 3.7). To remove excess de-PG2BAH-proK from the microchannel walls, the buffer plug was injected through the front inlet from the substrate syringe at a flow rate of 5 μL/min. Upon complete injection of the buffer plug, BODIPY-FL casein entered the chip through the front inlet (20 μg/mL, pH = 7.0) under steady state conditions at a flow rate of 14 nL/min. The activity of proK was then measured by fluorescence imaging. As proK coating of the microchannel walls took place downstream of the side inlet (see section 3.7), the proK activity, hence fluorescence signal, was determined in the marked region. 25973


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Figure 4. In situ UV/vis spectrophotometric monitoring of the conjugation reaction of de-PG2-HyNic and proK-4FB. (a) Changes of the absorbance A occurring between λ = 250 and 500 nm during the conjugation reaction; (b) time-dependent changes of the difference spectrum, as calculated from the spectra shown in panel a; (c) evolution of the characteristic BAH absorption around 354 nm during the first 5 h after starting the conjugation reaction (afterward, the increase in A354 was negligible). [HyNic] = 100 μM; [4-FB] = 50 μM.

3. RESULTS AND DISCUSSION 3.1. Preparation of ProK-4FB. ProK was modified in aqueous solution at pH = 8.0 with N-succinimidyl 4formylbenzoate (S-4FB, Figure 2) at various ratios of [proK]/[S-4FB] to yield a molar substitution ratio (MSR) of ≈1 or 410 nm = 0.00), i.e., no indication of scattering of visible light. The enzymatic activities of dissolved proK, modified proK (proK-4FB), and the denpol−proK conjugate (de-PG22000BAH-proK140) were determined at pH = 7.0 and T = 25 °C with Suc-AAPF-pNA as the substrate. All three proK samples obeyed Michaelis−Menten kinetics because the initial velocity vs initial Suc-AAPF-pNA concentration data (Figure 5) could

unmodified proK. A decrease in kcat can be explained by modifications of surface exposed lysine residues that are localized in the vicinity of the active site. Fortunately, none of the eight Lys of proK is localized very close to the active site (Figure S5). In the case of the conjugate de-PG22000-BAH-proK140, the determined KM and kcat values are clearly lower than the ones of unmodified proK, 66 ± 7 μM (KM) and 23 ± 1 s−1 (kcat), see Table 1. A decrease in kcat could be due to the restricted mobility of the denpol-bound proK and/or a hindrance, or complete blockage, of the substrate access to the active site of some of the proK molecules. In the latter case, the concentration of accessible active sites would be lower than the concentration of proK as determined through the quantification of the BAH bonds. The observed lower KM value for proK in the conjugate, as compared to free proK or proK-4FB, may be due to unspecific interactions between the conjugate and Suc-AAPF-pNA, leading to higher local substrate concentrations. Although the reasons for the differences of the catalytic constants between proK and de-PG22000-BAH-proK140 can only be hypothesized, the experimental results clearly prove that the prepared de-PG22000-BAH-proK140 was still catalytically active if dissolved in aqueous solution. The catalytic efficiency of de-PG22000-BAH-proK140 (kcat/KM = 0.35 ± 0.05 μM s−1) was about 1/3 of the one determined for proK (0.93 ± 0.11 μM s−1) or proK-4FB (1.00 ± 0.17 μM s−1), see Table 1. If dissolved in aqueous solution at pH = 7.0 and stored for up to 60 days at 4 °C, all three samples, proK, prok-4FB, and dePG22000-BAH-proK140, had indistinguishable relative stabilities (Figure 6). These data show that the many free amino groups

Figure 5. Hydrolytic activity of native, unmodified proK, proK-4FB, and de-PG22000-proK140, determined with the chromogenic substrate Suc-AAPF-pNA at pH = 7.0 (10 mM MOPS) and T = 25 °C. The rate of p-nitroaniline formation was measured as a function of Suc-AAPFpNA concentration between 30 and 250 μM for a proK concentration of 5 μM (proK and proK-4FB) and 16.5 μM (de-PG22000-BAHproK140). The lines through the experimental points represent fits to the Michaelis−Menten equation (see Eqn. S1) for the evaluation of KM and kcat given in Table 1. The adjusted R2 values for the three fits are 0.996 46 (proK), 0.990 72 (proK-4FB), and 0.989 96 (de-PG22000BAH-proK140), respectively.

be fitted with the Michaelis−Menten eq (Eqn. S1) with adjusted R2 values above 0.98, see section 2 and Figure 5. The results are shown in Table 1, together with a literature value for

Figure 6. Stability of native proK, proK-4FB, and de-PG22000-BAHproK140 stored at 4 °C, pH = 7.0. The proK and proK-4FB solutions were stored at a concentration of 1 μM. The concentration of the dePG2-BAH-proK solution corresponded to 10 μM proK as determined from the UV/vis absorption of the BAH linker. The enzymatic activity measurements were performed at pH = 7.0 (10 mM MOPS buffer) using the 100× diluted storage solution and 500 μM Suc-AAPF-pNA as substrate.

Table 1. Kinetic Constants of proK, proK-4FB, and dePG22000-BAH-proK140 Obtained from the Nonlinear Fit of the Initial Velocities to the Michaelis−Menten Equation (See Figure 5) KM (μM) proK proK-4FB de-PG2-BAH-proK proK (lit.)a

185 138 66 230

± ± ± ±

14 15 7 20

kcat (s−1) 172 138 23 178

± ± ± ±

8 8 1 5

kcat (KM/μM−1·s−1) 0.93 1.00 0.35 0.782

± ± ± ±

0.11 0.17 0.05 0.090

of de-PG2 that were still present in the conjugate had no deleterious effects on proK under the conditions used. For all three samples, after 60 days, 40−45% of the initial enzymatic activity remained, as determined with Suc-AAPF-pNA. Experiments in which the activity of free proK (333 nM) was measured with a similar substrate, Suc-AAA-pNA at pH = 7.0 and 25 °C (10 mM MOPS buffer, 0.15 M NaCl, 1% DMF) showed that free de-PG22000 (up to a r.u. concentration of 1 mM) has no dramatic influence on the activity of proK. The KM and kcat values determined for proK and the substrate Suc-AAApNA in the absence and presence of the denpol de-PG22000 (30 μM r.u.) were 0.43 ± 0.03 mM (no denpol) vs 0.62 ± 0.01 mM

a

Literature values are from Georgieva et al., measured in 0.1 M Tris/ HCl buffer, pH 8.2, 5% DMF, at 25 °C.49

free proK determined under similar conditions (pH = 8.2).49 For dissolved, free proK, the values determined for the Michaelis constant, KM, and for the turnover number, kcat, were rather similar to the literature values, 185 ± 14 μM vs 230 ± 20 μM,49 and 172 ± 8 s−1 vs 178 ± 5 s−1.49 Both values for proK-4FB were a bit lower, 138 ± 15 μM (KM) and 138 ± 8 s−1 (kcat), but still quite similar to the values determined for 25975


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ACS Applied Materials & Interfaces (with denpol), and 1.45 ± 0.04 s−1 (no denpol) vs 1.83 ± 0.02 s−1 (with denpol). The literature values determined for the same substrate but under a bit different conditions (pH = 8.0, 25 °C, 50 mM Tris−HCl, 0.1 M NaCl, 5% DMF) are 0.88 mM (Km), and 1.1 s−1 (kcat).50 The decrease in activity seen for free proK as well as for de-PG22000-BAH-proK140, if stored in solution, might be the result of an autocatalytic digestion of proK.51 3.4. TInAS Measurements. The adsorption of de-PG22000BAH-proK140 on silicate surfaces was investigated by using the transmission interferrometric adsorption sensor, TInAS,38,39 see Figure 7. Upon pumping an aqueous solution of the

characterize the catalytic activity of the immobilized proK, microscopy glass coverslips were used as supports and the dePG22000-BAH-proK140 conjugate was adsorbed in the same way as elaborated for the quantification of the adsorbed mass by the TInAS measurements, see above. After adsorption of the denpol−enzyme conjugate on the coverslips that were deposited inside polypropylene reaction tubes, the coverslips were first washed with pH = 7.0 buffer solution to remove excess conjugate, see section 2. Then, the activity of the obtained immobilized proK was quantified by comparing the rate of hydrolysis of Suc-AAPF-pNA at pH = 7.0 and T = 25 °C with the corresponding hydrolysis rates for known amounts of native proK dissolved in the pH = 7.0 buffer solution (Figure S3b). Although it is clear that the kinetic constants of immobilized proK are likely to be different from the kinetic constants of dissolved proK, for a rough estimation of the surface concentration of active proK, however, it was assumed that the kinetic constants are the same for the immobilized and for the free proK. With this assumption and with a calibration curve obtained with 0.1−2.0 nM proK in pH = 7.0 solution and the substrate Suc-AAPF-pNA (500 μM, Figure S3b), the apparent surface concentration of proK on the glass coverslips, Γapp, was determined as 2.0 pmol/cm2. This value is lower than the one obtained from the TInAS measurements (4.9 pmol/ cm2, see above). The reason for the difference between the two determinations may not only be due to the assumptions made with respect to the kinetic constants, as just mentioned, but also due to a direct contact of some of the proK molecules of adsorbed de-PG2-BAH-proK with the glass coverslip surface. Such interaction may lead to an inactivation of proK or to a decreased accessibility of the substrate molecules to the active site of proK. In any case, there is no doubt that the immobilized proK was still active against Suc-AAPF-pNA. In addition to the determination of the catalytic activity of the immobilized proK, the stability of the immobilized proK during storage was also investigated. The glass coverslips were stored in buffered solution at pH = 7.0 and at T = 4 °C, and the residual activity was determined by repeated activity measurements. In an initial phase (5 days), the activity dropped from Γapp = 2.0 pmol proK/cm2 to Γapp ≈ 1.0 pmol/cm2 (Figure 8). During further storage, the proK activity remained remarkably constant with a reduction to a level corresponding to Γapp = 0.8 pmol/cm2 after a storage time of 7 weeks (Figure 8). In control

Figure 7. TInAS analysis of the adsorption of de-PG22000-BAHproK140 from an aqueous solution at 14 μM r.u. on a SiO2 surface at pH = 7.0 (10 mM MOPS buffer). Formation of a stable layer on the surface with an adsorbed mass of about 200 ng/cm2 was observed. At this level, no further adsorption was observed, indicating a controlled adsorption of a thin layer. No desorption of the conjugate was observed during a subsequent flow of buffer solution (pH = 7.0, 10 mM MOPS) through the flow cell.

denpol−enzyme conjugate (14 μM r.u., pH = 7.0, 10 mM MOPS buffer) through the device, a stable layer of de-PG22000BAH-proK140 formed on the sputtered SiO2 surface within 30− 40 min at the chosen flow rate of 20 μL/min. With the estimated value for the refractive index increment for de-PG2BAH-proK, dn/dc = 0.173 cm3/g (see section 2), the adsorbed mass was calculated to be about 200 ng conjugate per cm2 (Figure 7). This level remained constant even after injection of a pH = 7.0 buffer solution at a flow rate of 0.4 mL/min to remove excess conjugate from the flow cell. No significant desorption of de-PG22000-BAH-proK140 was observed during this rinsing of the cell, indicating a stable adherence of the conjugate to the silicate surface, like in the case of de-PG21400BAH-HRP 108 and de-PG2 1400 -BAH-GOD ≈50 , 17 free dePG21400,17 or de-PG22000.44 Knowing the mass fraction of proK in de-PG22000-BAH-proK140, i.e., the calculated mass of proK in the conjugate divided by the total conjugate mass (=0.71), and knowing the molar mass of proK (=28 930 Da),47,48 the surface concentration of proK at saturation (200 ng/cm2, Figure 7) was calculated: 4.9 pmol proK/cm2. Because the adsorbed mass for de-PG22000-BAH-proK140 (200 ng/cm2) was about the same as the adsorbed mass in the case of unmodified denpol, de-PG22000 (Figure S6), we conclude that the silicate surface was not so densely covered with the conjugate, and that there were still some uncoated areas. Most importantly, however, the adsorbed conjugate layer remained strongly adsorbed, i.e., there was no desorption after washing with buffer solution (Figure 7). 3.5. Activity and Stability of de-PG22000-BAH-proK140 Immobilized on Microscopy Glass Coverslips. To

Figure 8. Storage stability of de-PG22000-BAH-proK140 adsorbed on a microscopy glass coverslip. The measured catalytic activity per surface area was correlated to an apparent surface concentration of the enzyme (Γapp) by comparison with a calibration curve obtained with native proK in solution. Native proK and a mixture of nonmodified dePG22000 and native proK adsorbed under the same conditions resulted in low levels of proK activity, accompanied by desorption from the surface. 25976


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operating conditions in the continuous flow setup, the system was run at room temperature at a constant flow rate of 35 μL/ min for 33 h and the product formation was continuously monitored. UV/vis absorption spectra recorded at an interval of 10 min indicated excellent stability of proK under these continuous flow conditions with a decrease of the rate of product formation of only 5% within 30 h (Figure 9). The remarkably low level of activity loss during operation was attributed to the very stable adsorption of the denpol− enzyme conjugate on the inner wall of the micropipette, with no loss of enzyme from the surface. In comparison, Slováková et al.32 determined the operational stability of a proK reactor prepared by covalent attachment or proK to magnetic particles. In this setup, a decrease of activity of 50% of the initial activity was found after 8 batch experiments of 15 min.32 3.7. Immobilization of de-PG22000-BAH-proK140 on a Microfluidic Chip. After the promising results obtained about the immobilization of proK inside a glass micropipette, the possibility for an application of the same de-PG22000-BAHproK140 conjugate for the immobilization of proK on a microfluidic chip was investigated. A PDMS chip with meander channel geometry was prepared and bonded on a glass coverslip. The channel was prepared such that it was connected to two inlets, one at the beginning (front inlet) and one in the middle of the meander (side inlet), see section 2 and Figure 10a. Injection of a pH = 7.0 buffer solution containing dePG22000-BAH-proK140 through the side inlet was used for the immobilization of proK on the glass coverslips in the area of the channel between the side inlet and the outlet. After adsorption of the denpol−enyzme conjugate, excess conjugate was

experiments using glass coverslips exposed to a solution containing free proK or a mixture of free proK and dePG22000 instead of de-PG22000-BAH-proK140, low levels of adsorbed proK were detected in the initial activity measurements. During storage, these levels dropped within 2 days to the background level of the enzymatic activity assay due to desorption from the surface (Figure 8). These results clearly demonstrate the suitability of the enzyme immobilization approach for the preparation of devices that are constituted of silicate surfaces and which are based on the proteolytic activity of immobilized proK. Even though the initial decrease of Γapp of proK was considerable, the subsequent very slow loss of activity during 7 weeks storage of the glass coverslips in solution indicates the potential of a prolonged storage in a wet state of devices prepared by simple adsorption of a de-PG2-BAH-proK conjugate. 3.6. Activity and Stability of de-PG22000-BAH-proK140 Immobilized Inside Glass Micropipettes. To apply the developed immobilization system for proK in a simple device for operation under continuous flow conditions, silicate glass micropipettes were used as support for the adsorption of dePG22000-BAH-proK140. The inner surface of a glass micropipette was coated with de-PG22000-proK140 by simply filling the micropipette with a dilute solution of the conjugate as used for the TInAS measurements (see section 3.4) and as used for the coating of the glass coverslips (see section 3.5). After the glass micropipette was rinsed with pH = 7.0 buffer solution (10 mM MOPS) to remove excess conjugate, the prepared flow reactor was installed in a setup for online quantification of the enzyme activity by UV/vis spectrophotometry (Figure 9). One end of

Figure 9. Operational stability of de-PG22000-BAH-proK140 adsorbed inside a silicate glass micropipette under continuous flow conditions. After 30 h, about 95% of the initial activity was maintained. Hydrolysis of Suc-AAPF-pNA (100 μM, pH = 7.0) was quantified at λ = 410 nm and the baseline shift was corrected by subtraction of the absorbance at 700 nm where no absorption band was present. The outliers were caused by air bubbles in the flow cell of the UV/vis spectrophotometer.

Figure 10. Proteolytic activity of the adsorbed de-PG22000-BAHproK140 conjugate in a microfluidic channel (width 100 μm; height 40 μm). (a) Conjugate for the proK immobilization on the chip was introduced at a proK concentration of 2 μM through an inlet in the 6th serpentine. After 1 h, the conjugate solution was removed from the chip by purging of the channel with a 10 mM MOPS buffer plug (pH = 7.0) through the front inlet. (b) After injection of the BODIPY-FLcasein substrate solution (20 μg/mL, pH = 7.0), an increase in fluorescence was observed with increasing distance from the side inlet, indicating proteolytic cleavage of the BODIPY-FL-casein due to the presence of immobilized de-PG22000-BAH-proK140. (c) Intensity profile across the channel segments shown in panel b.

the micropipette was connected with PTFE tubing to a reservoir containing a solution of Suc-AAPF-pNA (100 μM in pH = 7.0 buffer solution), whereas the other end was connected to a Z-flow cell of the UV/vis spectrophotometer and a peristaltic pump (Figure 9). At a constant flow rate, which was controlled by the peristaltic pump, the formed p-nitroaniline product was measured and found to exhibit a steady state. For investigating the stability of the immobilized proK under 25977


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useful because it is not only composed of long linear polymer chains that have an average length of about 500 nm (calculated for a stretched chain of 2000 r.u.), but it also has many functional groups (primary amines/ammonium ions) along the polymer chain due to the dendritic nature of the repeating units (8000 amines/ammonium ions in a 2000 r.u. long chain). The amines fulfill three purposes: (i) they serve in their neutral form as nucleophiles for the covalent binding of the enzymes to the polymer chain under mild reaction conditions in aqueous solution for obtaining the denpol−enzyme hybrid structure;17,36,37 (ii) they provide in their protonated state (as ammonium ions) water solubility in neutral or acidic aqueous media; and (iii) they are, at least partially, responsible for the binding of the conjugate to the silicate surfaces (though multiple interactions between the conjugate and the surface).44 Obviously, the noncovalent binding of the conjugate to silicate surfaces not only involves the denpol part of the conjugate, but also the proK molecules which were attached along the denpol chain (pI = 8.9),48 as schematically shown in Figure 1. Another aspect which has not been explored yet, but is relevant for real applications involving biological analyte solutions, is the potential antifouling properties of the polycationic de-PG2. From literature it is known that hyper-branched polycationic poly(ethylenimine), PEI, brushes have protein-resistant properties which are alike to the ones of the well-known poly(ethylene glycol), PEG.53 Whether de-PG2 has similar properties is not yet known. From a more practical point of view, there are no conceptual difficulties to apply our enzyme immobilization methodology to the immobilization of other types of enzymes as well, once a dendronized polymer like de-PG2 has been synthesized.54 The only steps that require optimization for each new type of enzyme of interest are (i) the initial ratio of S-4FB to enzyme and the optimal pH value for the enzyme modification reaction for achieving MSR < 1.0, and (ii) the purification of the conjugate obtained by repetitive ultrafiltration (e.g., proK or HRP)17 or ammonium sulfate precipitation (e.g., GOD).17 For all three de-PG2-BAH−enzyme conjugates prepared and tested so far, a strong adhesion of the conjugates to silicate surfaces was very evident, as well as the retention of the catalytic activities of the enzymes. Although the long-term stability of the immobilized enzymes needs to be tested for each enzyme of interest and is expected to depend on the enzyme type, we are convinced that this novel enzyme immobilization methodology for the preparation of miniaturized flow reactor systems is a valuable alternative to the existing ones54−57 and deserves a critical testing in real applications, such as sample pretreatment or purification.

removed from the channel by injection of a pH = 7.0 buffer plug though the front inlet. To monitor the proteolytic activity of proK inside the microfluidic channel by fluorescence microscopy, the fluorogenic substrate BODIPY-FL-casein was used. This casein derivative is labeled with multiple fluorescent BODIPY-FL tags. In native casein molecules bearing the tags, the fluorescence is quenched due to their spatial proximity. Upon proteolytic digestion, fragmentation of casein occurs which is accompanied by a corresponding increase in fluorescence.52 After injection of a solution of BODIPY-FL-casein (20 μg/mL, pH = 7.0) into the front inlet at a continuous flow rate of 14 nL/min, a constant background fluorescence was detected in the channel between the first and the second inlets (no denpol−enzyme conjugate present). In contrast to this, in the area of the channel containing adsorbed de-PG22000-BAHproK140 after the second inlet, an increasing fluorescence was observed. Under steady state conditions, the fluorescence level within the channel increased with increasing distance from the second inlet, indicating a proteolytic digestion of BODIPY-FLcasein (Figure 10b,c). At an elevated flow rate of 1 μL/min, no increase in fluorescence was observed in the area of the channel where the denpol−enzyme conjugate was present. This indicates that in these experiments the residence time of BODIPY-FL-casein was too short for observing a significant, i.e., detectable hydrolysis. Control measurements were carried out by using the denpol only, de-PG22000, instead of de-PG22000-BAH-proK140. In these experiments, no increase in fluorescence was observed after the second inlet (Figure S7), thereby clearly demonstrating that the increase in fluorescence inside the microfluidic channels was due to the presence of proK acting on the fluorescently labeled casein substrate. All in all, the experiments that were performed demonstrate that the elaborated immobilization methodology can be applied successfully for the simple preparation of microfluidic chips containing immobilized proK in catalytically active state.

4. CONCLUSIONS Through the preparation of a hybrid structure consisting of the dendronized polymer de-PG22000 and several copies of proK, we have shown that the immobilization of proK on silicate surfaces is possible in a rather straightforward and highly reproducible way. Simple adsorption of the conjugate de-PG22000-BAHproK140 from a dilute aqueous solution resulted in a noncovalent, but very stable, coating of silicate surfaces with the denpol−proK conjugate. The enzyme is active and has a high operational stability if analyzed in an easily prepared glass micropipette flow reactor, as tested with a small chromogenic substrate (Suc-AAPF-pNA). What remains to be elaborated is the efficiency of the immobilized proK toward macromolecular substrates because possible applications of immobilized proK are in the field of proteomics23−27 and prion protein research28,29 where proteins are the substrates of interest. First measurements with a casein derivative (BODIPY-FLcasein, Figure 10) indicated that the immobilized proK is indeed active toward proteins. Investigations with other proteinaceous substrates are in progress. From a more general point of view, the work carried out demonstrates that the methodology developed previously for the immobilization of the two oxidative enzymes HRP and GOD17 can also be applied successfully for proK as proteolytic model enzyme. The denpol de-PG22000 turned out to be very

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b09301. Schemes for the preparation of de-PG2-HyNic and dePG2-BAH-proK; details about proK and proK activity measurements; a TInAS analysis of de-PG22000; and control experiments with the microchip (PDF).

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AUTHOR INFORMATION

Corresponding Author

*P. Walde. E-mail: [email protected]. Tel: +41 44 632 0473. 25978


Research Article

ACS Applied Materials & Interfaces Author Contributions

(13) Palazzo, G.; Colafemmina, G.; Guzzoni Iudice, C.; Mallardi, A. Three Immobilized Enzymes Acting in Series in Layer by Layer Assemblies: Exploiting the Trehalase-Glucose Oxidase-Horseradish Peroxidase Cascade Reactions for the Optical Determination of Trehalose. Sens. Actuators, B 2014, 202, 217−223. (14) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Threedimensional Structures of Avidin and the Avidin-Biotin Complex. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 5076−5080. (15) Derewenda, Z.; Yariv, J.; Helliwell, J. R.; Kalb (Gilboa), A. J.; Dodson, E. J.; Papiz, M. Z.; Wan, T.; Campbell, J. The Structure of the Saccharide-binding Site of Concanavalin A. EMBO J. 1989, 8, 2189− 2193. (16) Jesionowski, T.; Zdarta, J.; Krajewska, B. Enzyme Immobilization by Adsorption: a Review. Adsorption 2014, 20, 801−821. (17) Küchler, A.; Adamcik, J.; Mezzenga, R.; Schlüter, A. D.; Walde, P. Enzyme Immobilization on Silicate Glass Through Simple Adsorption of Dendronized Polymer-Enzyme Conjugates for Localized Enzymatic Cascade Reactions. RSC Adv. 2015, 5, 44530− 44544. (18) Gustafsson, H.; Küchler, A.; Holmberg, K.; Walde, P. Coimmobilization of Enzymes with the Help of a Dendronized Polymer and Mesoporous Silica Nanoparticles. J. Mater. Chem. B 2015, 3, 6174−6184. (19) Grotzky, A.; Manaka, Y.; Kojima, T.; Walde, P. Preparation of Catalytically Active, Covalent α-Polylysine-Enzyme Conjugates via UV/Vis-quantifiable Bis-aryl Hydrazone Bond Formation. Biomacromolecules 2011, 12, 134−144. (20) Hermanson, G. T. Bioconjugate Techniques, 3rd ed.; Elsevier: Amsterdam, 2013. (21) Betzel, C.; Pal, G. P.; Saenger, W. Three-Dimensional Structure of Proteinase K at 0.15-nm Resolution. Eur. J. Biochem. 1988, 178, 155−171. (22) Mathew, C. G. P. The Isolation of High Molecular Weight Eukaryotic DNA. In Meth. Mol. Biol., Vol. 2: Nucleic Acids; Walker, F. M., Ed.; Humana Press: New York, 1984; pp 31−34. (23) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R., III A Method for the Comprehensive Proteomic Analysis of Membrane Proteins. Nat. Biotechnol. 2003, 21, 532−538. (24) Liuni, P.; Rob, T.; Wilson, D. J. A Microfluidic Reactor for Rapid, Low-Pressure Proteolysis with On-chip Electrospray Ionization. Rapid Commun. Mass Spectrom. 2010, 24, 315−320. (25) Liu, S.; Bao, H.; Zhang, L.; Chen, G. Efficient Proteolysis Strategies Based on Microchip Bioreactors. J. Proteomics 2013, 82, 1− 13. (26) Li, Y.; Yan, L.; Liu, Y.; Qian, K.; Liu, B.; Yang, P.; Liu, B. Highefficiency Nano/Micro-Reactors for Protein Analysis. RSC Adv. 2015, 5, 1331−1342. (27) Cheng, G.; Hao, S.-J.; Yu, X.; Zheng, S.-Y. Nanostructured Microfluidic Digestion System for Rapid High-performance Proteolysis. Lab Chip 2015, 15, 650−654. (28) Soto, C. Diagnosing Prion Diseases: Needs, Challenges, and Hopes. Nat. Rev. Microbiol. 2004, 2, 809−819. (29) Le Nel, A.; Minc, N.; Smadja, C.; Slovakova, M.; Bilkova, Z.; Peyrin, J.-M.; Viovy, J.-L.; Taverna, M. Controlled Proteolysis of Normal and Pathological Prion Protein in a Microfluidic Chip. Lab Chip 2008, 8, 294−301. (30) Merck GmbH. Immobilized Enzymes. Patent WO 94/22574, October 13, 1994. (31) Merck GmbH. Immobilisierte Proteinase K. Patent DE 43 33 213 A1, March, 30, 1995. (32) Slováková, M.; Peyrin, J.-M.; Bílková, Z.; Juklíčková, M.; Hernychová, L.; Viovy, J.-L. Magnetic Proteinase K Reactor as a New Tool for Reproducible Limited Protein Digestion. Bioconjugate Chem. 2008, 19, 966−972. (33) Eker, B.; Asuri, P.; Murugesan, S.; Linhardt, R. J.; Dordick, J. S. Enzyme-Carbon Nanotube Conjugates in Room-Temperature Ionic Liquids. Appl. Biochem. Biotechnol. 2007, 143, 153−163. (34) Sommer, C.; Hertel, T. C.; Schmelzer, C. E. H.; Pietzsch, M. Investigation on the Activation of Recombinant Microbial Pro-

The paper was written through contributions of all authors. All authors have given approval to the final version of the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Andrea Grotzky for the synthesis of S-HyNic, S4FB, and the denpol used in this work. We also acknowledge the MS service of the laboratory of organic chemistry at ETH (Louis Bertschi) for the analysis of the proK samples. The help by the following Bachelor students of Material Science at ETH (“Praktikum V”) with some of the experiments carried out is highly appreciated: Alina Hauser, Cristina Mercandetti, Christos Glaros, Livia Schneider, and Ines Weber.

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ABBREVIATIONS BAH, bis-aryl hydrazone denpol, dendronized polymer de-PG2, deprotected dendronized polymer second generation GOD, Aspergillus sp. glucose oxidase HRP, horseradish peroxidase isoenzyme C PDI, polydispersity index PDMS, polydimethylsiloxane PG2, dendronized polymer second generation proK, proteinase K r.u., constitutional repeating units

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