Microfluidic Reactors Based on Rechargeable Catalytic Porous

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Microfluidic Reactors based on Rechargeable Catalytic Porous Supports: Heterogeneous Enzymatic Catalysis via Reversible Host-Guest Interactions Alberto Sanz de León, Nelson Vargas Alfredo, Alberto Gallardo, Alfonso FernándezMayoralas, Agatha Bastida, Alexandra Munoz-Bonilla, and Juan Rodriguez-Hernandez ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13554 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on December 31, 2016

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ACS Applied Materials & Interfaces

Microfluidic Reactors based on Rechargeable Catalytic Porous Supports: Heterogeneous Enzymatic Catalysis via Reversible Host-Guest Interactions

Alberto Sanz de León,1,4 Nelson Vargas-Alfredo,1 Alberto Gallardo,1 Alfonso Fernández-Mayoralas,2 Agatha Bastida,2 Alexandra MuñozBonilla1,3 and Juan Rodríguez-Hernández1*

1

Instituto de Ciencia y Tecnología de Polímeros (ICTP), Consejo Superior de

Investigaciones Científicas (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain. Email: [email protected] 2

Instituto de Química Orgánica General, (IQOG-CSIC), Juan de la Cierva 3, 28006,

Madrid, Spain. 3

Departamento de Química-Física Aplicada, Facultad de Ciencias, Universidad

Autónoma de Madrid, C/Francisco Tomás y Valiente 7, Cantoblanco, 28049 Madrid, Spain. 4 Mechano(Bio)Chemistry, Max Planck Institute of Colloids and Interfaces, Science Park Potsdam-Golm, 14424 Potsdam, Germany

1 ACS Paragon Plus Environment


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ABSTRACT We report on the fabrication of a microfluidic device in which the reservoir contains a porous surface with enzymatic catalytic activity provided by the reversible immobilization of horseradish peroxidase onto micrometer size pores. The porous functional reservoir was obtained by the Breath Figures approach by casting in a moist environment a solution containing a mixture of high molecular weight polystyrene (HPS) and a poly(styrene-co-cyclodextrin based styrene) (P(S-co-SCD)) statistical copolymer. The pores enriched in CD were employed to immobilize horseradish peroxidase (previously modified with adamantane) by host-guest interactions (HRP-Ada). These surfaces exhibit catalytic activity that remains stable during several reaction cycles. Moreover, the porous platforms could be recovered by using free water soluble β-CD with detergents. An excess of β-CD/TritonX100 in solution disrupt the interactions between HRP-Ada and the CD-modified substrate thus allowing us to recover the employed enzyme and reuse the platform.

KEYWORDS Breath Figures, Host-Guest interactions, enzymatic catalysis, heterogeneous catalysis, reusable bioreactors, surface modification.


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INTRODUCTION

Microfluidic devices have found application in many different areas such as combinatorial synthesis, biosensing or analytical chemistry.1-3 Reducing the required volumes for a particular reaction has several important advantages including the waste reduction, better temperature control or improving the kinetics of diffusion-limited reactions.4 These are crucial advantages in the fabrication of microfluidic reactors for enzymatic catalysis. Enzymatic catalysis is currently a topic of considerable research interest due to several reasons.5-7 For instance, the unique secondary and tertiary structure of enzymes allow them to carry out specific reactions in which a define substrate can react while the rest of the molecules present in the media remains unaffected. Likewise, enzymes are able to catalyse reactions that cannot be achieved by any other organic or inorganic catalytic compounds resulting in excellent chemo-, regio-, and stereoselectivity

8-9

. Finally, reactions catalysed by enzymes occurs using mild reaction

conditions in water, an environmentally friendly solvent.10 However, enzymes exhibit limited solution stability since denaturation and, as a result, deactivation occurs upon a certain period of time.11-12 On the other hand, from the practical point of view, recycling and reuse of these enzymes require tedious and time-consuming separation and purification steps. In this context, enzyme immobilization has been proposed as an interesting alternative to overcome the above depicted issues. This strategy has been systematically employed during the last decades because immobilization stabilizes the enzyme structure and prevents from denaturation, reactions can be carried out in a continuous way and no purification processes are, thus, required.13-14 However,



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several aspects concerning enzyme immobilization still require further investigation. First of all, ideally immobilization should be carried out using mild reaction conditions in order to preserve the enzyme activity.15 Second, it is desirable to carry out the immobilization step onto substrates exhibiting large surface areas in order to increase the amount of enzyme immobilized.16-17 It would be desirable to have platforms able immobilize the enzyme for a particular reaction that could be, in turn, reused to immobilize other enzymes for another type of reaction. Moreover, the enzyme interaction with the substrate needs to be strong enough to prevent desorption but, at the same time, permits the enzyme mobility and conformational changes required to carry out the chemical reaction. This balance is quite related to the immobilization mode. A number of immobilization techniques and strategies to elaborate solid supports have been reported during the last years. These strategies resort to the use of either physical or covalent immobilization. Physical immobilization leads in most cases, to an enzyme release

18

and thus a loss of activity. Covalent immobilizations, which are much

more stable, usually result in a loss of enzyme activity, as mentioned above.19 In addition, covalent anchoring limit the surfaces produced to one single use. Herein, we present the fabrication of a microfluidic lab-on-a-chip device that will take advantage of the surface immobilization of adamantane modified enzymes via host-guest interactions with a cyclodextrin modified surface. Host-guest interactions are, in comparison with the above mentioned strategies of enzyme immobilization, an interesting alternative since the interactions formed are strong enough to prevent enzyme release and they can be displaced and re-constructed by using the appropriate molecules.


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In addition to the reversible immobilization, in this manuscript, we will carry out then functionalization of the device by using the breath figures approach, which renders in a single step a large surface porous area and an inner functionalization of the pore with CD moieties. This selective functionalization is achieved by using polymer blends as precursors. As will be depicted, this methodology permits the fabrication of functional porous films with narrow pore size distribution, is fast and does not require of expensive equipment. Finally, the use of polymer blends as precursors will allow us to precisely localize the enzyme within the cavities in which the liquid speed flow is reduced favouring the contact between the enzyme and the substrate. In addition, this reversible immobilization on porous substrates has been carried out on a reduced volume microfluidic reactor manufactured using a stereolithographic (SLA) printing technology.

EXPERIMENTAL SECTION Materials High molecular weight polystyrene (Aldrich, Mw =2.50·105 g/mol) was used as polymeric matrix. Tetrahydrofuran (THF) was obtained from Alfa Aesar. Chloroform (CHCl3), dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), dichloromethane, dichloroethane, hexane, ethyl acetate and ethanol (EtOH) were purchased from Scharlau. 2,2’-Azobis-isobutyronitrile (AIBN, Merck) was recrystallized twice from ethanol. Styrene (Aldrich) was distilled before use. Adamantane-1-carbonyl chloride was obtained from Alfa Aesar, and the rest of chemicals required were provided by Aldrich. Potassium phosphate monobasic and dibasic, β-cyclodextrin (CD), 2,2’-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide (30 % w/w) and horseradish peroxidase Type VI-A (HRP) enzyme were purchased from Sigma and used



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as received. Bradford Protein Assay from Bio-Rad was used for measuring the soluble protein concentration at 595nm.

Methods NMR (1H and 13C) spectra were recorded on a Bruker Avance III HD-400 spectrometer. High resolution mass spectra (HRMS) were recorded on an Agilent 6520 Accurate Mass Q-TOF spectrometer with an ESI source. Characterization of the surfaces was performed by scanning electron microscopy (SEM) using a Philips XL30 with an acceleration voltage of 25 kV. The samples were coated with gold-palladium (80/20) prior to scanning. Data processing of the SEM images recorded was carried out with ImageJ & AxioVision SE64 Rel. 4.9.1 software. Polymer synthesis Polystyrene bearing side β-CD moieties has been prepared by facile standard radical copolymerization of styrene (S) and a monomer modified with cyclodextrin (SCD) at 10 weight % of SCD with respect to S. SCD is a styrenic monomer previously described in the literature 20. This P(S-co-SCD) copolymer was obtained using previously reported procedures.21 Example of recipe: 115 µL of S (104 mg), 10.4 mg of SCD and 2.5 mg of AIBN were dissolved in 1 mL of DMF:DMSO 1:1. The total concentration of comonomers was 1 mol/L and the initiator concentration was 0.015 mol/L. Reactions were carried out in the absence of oxygen by gently bubbling nitrogen for 20-30 min before sealing the system. Polymer was isolated by exhaustive dialysis against dioxane followed by lyophilisation. MnSEC = 6800 g/mol, PDSEC = 1.50.


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Preparation of porous polymeric surfaces with pores enriched in cyclodextrin (CD). In a typical breath figures method, blends containing 10, 20 and 50 wt% of the copolymer P(S-co-SCD) previously synthesized and HPS matrix were dissolved in a solution with a total concentration of 30 mg/mL. THF or a mixture of THF:CHCl3 (1:4 or 1:1 v/v) were employed as solvents. Then, this solution was drop-cast onto 1,2 cm in diameter round glass coverslips (Ted Pella Inc.) inside a closed chamber with saturated relative humidity (RH) at room temperature. Also, as control, surfaces with 100 wt% HPS were fabricated in the same conditions.

Synthesis of N-Adamantane-1-carbonyl fluoresceinamine (Ada-FAm) According to our previously reported procedure21, to a solution of adamantane-1carbonyl chloride (12 mg, 0.06 mmol) in dry dichloromethane (0.3 mL) was added a mixture of fluoresceinamine (15 mg, 0.043 mmol), trimethylamine (9.3µL, 0.06 mmol) and 4-dimethylpyridine (1 mg) in dry dichloroethane (0.3 mL). The reaction mixture was stirred for 30 min at 0 ºC and for 3 h at room temperature. After this time, the solvent was evaporated and the residue was purified by flash chromatography (ethyl acetate - hexane, 5:1 → 4:1) obtaining N-adamantane-1-carbonyl fluoresceinamine (10.4 mg). 1H NMR (400 MHz, CD3OD) δ 7.03 (d, J = 2.1 Hz, 1H, Ar), 6.92 – 6.86 (m, 2H, Ar), 6.71 (d, J = 8.7 Hz, 2H, Ar), 6.62 (dd, J = 8.7, 2.3 Hz, 1H, Ar), 6.57 (d, J = 2.4 Hz, 1H, Ar), 6.54 (d, J = 8.7 Hz, 1H, Ar), 6.43 (dd, J = 8.7, 2.4 Hz, 1H, Ar), 1.90 (s, 9H, adamantyl), 1.71 – 1.53 (m, 7H, adamantyl). 13C NMR (101 MHz, CD3OD) δ 175.74, 170.78, 159.59, 152.42, 152.40, 151.99, 150.34, 141.22, 128.82, 128.65, 127.70, 124.11, 122.33, 117.43, 117.18, 112.33, 110.42, 109.80, 107.18, 102.16, 40.92, 38.72,



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38.41, 36.24, 36.06, 28.07, 27.94. HRMS (ESI) m/z calcd for C31H27NO6, 509.1838; found, 510.1896 [M +H]+.

Synthesis of the HRP-Ada HRP modified with adamantane moieties (HRP-Ada) were synthesized following previously reported procedures 22. For this purpose, a reaction mixture containing HRP (10 mg), 1-adamantanecarboxylic acid (10 mg) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 10 mg) was stirred for 1 h at room temperature (28 ºC) and then for 16 h at 4 ºC in a 100 mM sodium phosphate buffer at pH 6.0 (5 mL). The solution was further dialyzed against 50mM sodium phosphate buffer pH 7.0. In order to evidence that the modification step does not completely denature the enzyme, the activity of the HRP-Ada was measured with ABTS as described below. Fabrication of the microfluidic reactor The 3D microreactor was designed using Autodesk Inventor 2015 and manufactured using a stereolithographic (SLA) printing technology. For that purpose a Project 1200 3D printer from 3D systems was employed. The resolution achieved was 30 µm in z and around 56 µm (effective 585 dpi) in XY. VisiJet® FTX Clear material with a transparent clear appearance was employed as photosensitive resin. The smallest channel feature that has been produced using this material is 50 µm. Enzyme immobilization and quantification of enzyme adsorbed The films were immersed in 0.32 mL of a potassium phosphate buffer 40 mM pH 6.8 solution of HRP-Ada (0.01 mg/mL) for 4 h. At different times the supernatant solution was measured at 595nm with Protein assay (Bio Rad) reactive to determinate the protein concentration not adsorbed. Films were then rinsed with water, 40mM potassium phosphate pH 6.8 and 40mM potassium phosphate with 0.25% (w/v) bovine serum


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albumin and 0.5% (v/v) Triton X-100 at 25º. As control, blank experiments were carried out using either HPS surfaces without β-CD moieties and unmodified HRP enzyme (0.01mg/mL) to evaluate the presence of non-specific interactions.

Measurement of enzymatic activity The fabricated microfluidic reactor has been employed in a batch mode. Activity of the immobilized peroxidase enzyme (HRP from horseradish Type VI-A) was followed by a spectrofluorometric assay using 2,2’-Azido-bis(3-Ethylbenzthiazoline-6-sulfonic acid as a substrate (ABTS,ε = 36.8 mM-1 cm-1).So, 0.32 mL of a phosphate buffer solution (40 mM pH 6.8) containing ABTS (0.91 mM) were incubated onto the surfaces (HRPHPS) together with H2O2 (0.011 mL at 0.03 %wt). Periodically the supernatant was transfer to a cuvette to monitor by UV at λ=405 nm (Figure 4) the formation of a greencolored solution (ABTS+ cation). After that the solution was transfer back to the surfaces to let the reaction procced. The same protocol was carried out using HPS surface without CD moieties, as control, no detecting green color.

Enzyme release Release of HRP-Ada from HPS + P(S-co-SCD) surfaces was performed using a potassium phosphate 40 mM pH 6.8 buffer solution with 0.2% Triton X100 and 3mg/mL of albumin and containing an excess (5.0 mg/mL) of free β-CD for 2 h at 25ºC. The surfaces were then rinsed using the same buffer. Finally, the enzymatic activity of the surface was evaluated. Equally, the protein concentration released in the solution was determined by the Bradford protein assay.

RESULTS AND DISCUSSION



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Design of the microfluidic reactor An illustrative scheme of the fabrication of the microfluidic device and of the incorporation of the functional porous surfaces is presented in Figure 1. By means of a computer-aided design (CAD) the simplest fabricated microreactor consists of an inlet and outlet small reservoirs connected by a 100 µm diameter channel (a). In the center of the microreactor a support has been fabricated in which the porous functional material (supported in a round thin glass) will be placed (b). This configuration will permit a real time analysis of the catalytic reaction. Finally, a transparent cover (also prepared using the visijet clear material) that allows us to introduce the chemical substrates for the catalytic reaction was constructed and placed on top of the device (c). Porous film

(a)

Top view

(b) 100µm channel

Cover

Top view 2mm

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(c)

Cross-section

Inlet

Outlet

1,2 cm

Figure 1. Scheme of the 3D printed microreactor and the incorporation of the porous functional material. (a) Illustration of the top view and cross-section of the 3D printed microchip, (b) placement of the porous film in the center of the microchip and (c) closed system with a cover having an inlet and an outlet.

Preparation of the porous compartments 10 ACS Paragon Plus Environment

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The polymeric microporous surfaces were prepared by the breath figures method by solvent casting inside a closed chamber with a relative humidity above 90% at room temperature. The polymeric solution containing both the polymer matrix, i.e. high molecular weight polystyrene (HPS) and the functional amphiphilic copolymer i.e. polystyrene-co-poly(styrene-cyclodextrin) P(S-co-SCD) was deposited onto pre-cleaned glass coverslips and allowed to evaporate. In particular, we focused on blends of HPS and 10, 20 and 50 wt% of P(S-co-SCD). For comparative purposes, an additional sample prepared using exclusively HPS was fabricated. THF or a mixture of THF:CHCl3 (1:4 or 1:1 v/v) were employed. For all the compositions, a total concentration of polymers was set to 30 mg/mL. As depicted in Figure 2, the use of THF as single solvent leads to rather disordered surface patterns as well as using a mixture enriched in CHCl3 (THF:CHCl3 1:4 v/v). In contrast the use of a mixture of THF:CHCl3 1:1 v/v significantly improved the pore regularity and their distribution. CHCl3, extensively employed to produce breath figures, did not easily dissolved the copolymer bearing β-CD units probably affecting the homogeneity of the patterns observed. On the other hand, THF produced microporous films with rather disordered surface patterns. As a result, the combination of these two solvents allowed us to produce micropatterned films with improved pore regularity (Figure 2). Concerning the blend composition, it is possible to observe that an increase of the amount of copolymer resulted both in a decrease of pore average diameter and a decrease of the pattern regularity. Thus, whereas pores with sizes ~ 6,5 µm were observed when 10 wt% of P(S-co-SCD) was employed, diameters around 3,1 µm were measured in the case of 50% of P(S-co-SCD). The following



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immobilization, as well as the catalytic experiments, was carried out using the samples containing 10wt% of P(S-co-SCD) which lead to more regular patterns.

Figure 2. SEM images of the porous surfaces prepared from different blends. Above: Images of the films obtained using 10wt% of P(S-co-SCD) and 90wt% of HPS and THF, THF:CHCl3 (1:4 or 1:1) as solvent. Below: Porous films obtained from blends with higher amount 20wt% and 50wt% of P(S-co-SCD). (D: pore diameter, ID: interpore distance)

Localization of the cyclodextrin groups and enzyme immobilization Preparing films by the breath figures approach from polymer blends has several major advantages. For instance, the amount of functional polymer employed, which is usually more expensive and not commercially available, is reduced. 12 ACS Paragon Plus Environment

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Blending also allows for a controlled distribution of the functional polymer at the microporous surface. This is due to the reorganization phenomena occurring during the water condensation process.23-26 During this process, water droplets are formed at the interface and provided the presence of polar groups within the copolymer structure (as it is in our case the cylodextrin-containing SCD units of P(S-co-SCD)). These groups will tend to migrate toward the water interface stabilizing the droplets. As a result, once the solvent and condensed water droplets have been completely evaporated, these hydrophilic side groups are selectively located inside the pores and available for further surface modification processes. In order to provide evidence of the selective location of the β-CD groups inside the pores and their availability for further modification, we labeled adamantane-1-carbonyl chloride with fluoresceinamine (Ada-FITC) and carried out experiments to prove the presence of the β-CD moieties at the pore interface to establish host-guest interactions. In Figure 3 (center) are depicted the SEM images of the porous films prepared using the blends depicted above and those obtained using only HPS as control. In both cases, honeycomb patterned porous films with pore diameter of ~3 µm are obtained. Equally, Figure 3 shows the fluorescence images of the same films obtained upon interaction with Ada-FITC. As can be observed, whereas those films obtained from pure HPS did not exhibit fluorescence indicating that the Ada-FITC could not be immobilized onto the surface, those prepared using P(S-co-SCD) as additive show localized fluorescent signal within the pores. This result evidenced two important features of these surfaces. On the one hand, the presence of SCD moieties is a requirement to produce host-guest interactions with adamantane. On the other hand, these interactions are localized in the pore surface.

Planar surfaces prepared by



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decreasing the relative humidity did not produced any fluorescence indicating the absence of available cyclodextrin groups. Finally, it is worth mentioning that the experimental conditions employed produced, as depicted in Figure 3 a single layer of pores.

Figure 3. SEM micrographs and the corresponding fluorescence images of a) HPS and b) HPS + P(S-co-SCD) films obtained upon incubation with Nadamantane-1-carbonyl fluoresceinamine (Ada-FITC).

Catalysis inside the microreactor Once it has been proved that the cyclodextrin groups are available within the pores and they could establish interactions with adamantane groups, the enzyme was immobilized inside the cavities. In particular, the enzyme selected for this work, horseradish peroxidase binds to H2O2 forming a complex able to oxidize different compounds. In this case, azido-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was employed as model substrate. This compound, in its oxidized


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form presents a bluish green color, that can be easily detected by measuring UVVis at a fixed wavelength of λ = 405 nm (Figure 4 a-b). Prior to the immobilization step, the enzyme was firstly partially modified with adamantane (Ada) groups following previously reported procedures.22 The strategy involves the reaction between the amine groups of lysine aminoacids present at the enzyme surface and carboxylic acid groups of a modified adamantane molecule. This strategy permits, according to previous studies, to functionalize the enzyme while maintaining the secondary structure and its catalytic activity. In order to test the effect of the modification of the enzyme we explore the catalytic activity in solution of: the unmodified enzyme (HRP), the enzyme bearing adamantane moieties (HRP-Ada) and a control experiment without H2O2 (Control). Our experiments (depicted in Figure 4d) revealed that the activity in solution of the HRP upon modification with adamantane decreased maintaining, however, around 50% of the initial activity.



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Figure 4. a) Scheme of the oxidation of ABTS by interaction with HRP and H2O2, b) control of the reaction and c) green color observed as a result of the oxidation of ABTS, d) activity of the soluble HRP, HRP-Ada and its control (OD stands for Optical Density).

The modified enzyme was subsequently immobilized on the porous films via specific host-guest interaction. For this purpose, a solution of Phosphate Buffer (PB) 40 mM pH 6.8 containing 0.01 mg/mL of the modified enzyme (HRP-Ada) was incubated onto the porous surfaces for 60 min. Then, the solution was removed and the surface was extensively rinsed with the same buffer. To address whether this immobilization could also occur by non-specific interactions of the


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HRP with the HPS surfaces two additional control experiments were prepared using the same experimental conditions (depicted in Figure 5 a-c). On the one hand, a non-modified HRP was incubated onto a surface prepared using the functional copolymer P(S-co-SCD). On the other hand, modified HPR, i.e. HRPAda, was incubated onto a surface with only HPS. Moreover, the same volumes of ABTS and H2O2 were mixed in absence of any surface to check that the amount of H2O2 is low enough not to have a high chemical hydrolysis and be sure that the oxidation of ABTS is exclusively caused by the enzymatic hydrolysis. In order to understand the kinetics of the modified surfaces, the evolution of the absorbance was measured at different times for the samples depicted in Figure 5. In Figure 5d are depicted the evolution of the absorbance as a function of the reaction time. A remarkable and linear increase in the absorbance was only obtained in the case of Ada modified HRP onto P(S-co-SCD) surfaces. As observed in the image, only in this case the reservoir where the catalysis happens turns into green color after 1 h of incubation. In the rest of the cases an almost negligible catalytic activity can be observed. This residual catalytic activity can be attributed to the chemical hydrolysis of the substrate which, as we expected, can be neglected when compared to the enzymatic hydrolysis in the case where the host-guest interaction happens. The activity of the HRP-Ada supported was ~33 U/mg (Figure 5e).



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Figure 5. Immobilization of a) unmodified HRP onto HPS + P(S-co-SCD) surface; b) HRP-Ada onto HPS surface and c) HRP-Ada onto HPS + P(S-co-SCD) surface. In all cases the enzyme was incubated for 60 min with a concentration of 0.01 mg/mL in PB buffer 40 mM pH 6.8. d) Variation of the absorbance at 405nm during the catalytic oxidation of ABTS using substrates a)-c). e) Activity of the different substrates.

Stability of the enzyme immobilization and recovery evaluation Once we have proved the specificity of the host-guest interaction to immobilize only the enzymes bearing Ada moieties, two different questions required to be addressed. On the one hand, we explored the stability of the host-guest interactions and the enzymatic activity during repetitive cycles. On the other hand, the use of host-guest interactions permits the enzymatic recovery by using the appropriate conditions. To address whether the surfaces and in particular the host-guest interactions are strong enough to accomplish several reaction cycles we carried out the same catalytic assay onto a same surface for 5 cycles. Subsequently to each cycle, the 18 ACS Paragon Plus Environment

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surface was thoroughly rinsed with PB buffer. As it can be seen in Figure 6a, the surfaces present catalytic activity during the first three cycles explored (up to 5 in Figure 6(b)). More interestingly, the catalytic rate observed is stabilized during the cycles and exhibit an average enzymatic activity of 28 ± 5 U/mg

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Figure 6. a) Evolution of the absorbance measured during the first three catalytic cycles; b) Enzymatic activity measured for 5 consecutive cycles evidencing a constant activity of the surfaces.

Host-guest interactions can be formed and disrupted reversibly depending on the environmental conditions, such as the presence of a molecule with a higher affinity or the variation of the concentration of the guest molecule among others.27 In view of the potential use of these catalytic surfaces with other enzymes and taking advantage of the host-guest interactions to reuse these functional porous surfaces we evaluated the possibility to recover the immobilized HRP-Ada enzyme. For this purpose, a displacement of the immobilized HRP-Ada from the surface is attempted by means of an excess of free β-CD

21

. Surfaces containing HRP-Ada were immersed in an aqueous 19 ACS Paragon Plus Environment


solution of 6.0 mg/mL of β-CD in buffer phosphate pH=6.8 with 0.2% Triton X100 and 3mg/mL of albumin during 2 h at 25ºC. The supernatant was removed and then surfaces were rinsed several times. Afterwards, the catalytic assay was repeated using the same conditions. As depicted in Figure 7a, no catalytic activity could be observed in the surfaces exposed to β-CD. Therefore, we can conclude that the release of the HRP-Ada from the substrate has been successfully achieved (Figure 7b). Moreover, we can estimate, taking into account the activity of the released enzyme, that around 1-3µg of HRP enzyme were adsorbed in around 1 cm2.

Control Cycle 1 Upon HRP-Ada removal

1,2 0,9 0,6 0,3

30 20 10

20

40

60

Time (min)

80

a U p r e on H m ov R P al -A d

0

[P S+ P( + H S-c R P- o-S Ad C a D)]

0 C on tro l

0,0

(b) A (U/mg)

(a) 1,5 OD (405 nm)

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Page 20 of 25

Figure 7. a) Evolution of the absorbance at 405nm for control surfaces (red), surfaces with HRP in the first cycle (black) and upon removal from the surface using β-CD (green). b) Catalytic activity of the surfaces explored in a).

CONCLUSIONS


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In conclusion, we have presented here a new methodology to fabricate microfluidic reactors by producing reusable catalytic polymeric surfaces in a straightforward manner. For this purpose, the breath figures approach employed using a polymer blend leads to porous films with cyclodextrin units immobilized inside of the pore surface. Moreover, these groups were available to establish, via host-guest, interactions with adamantane groups. Thus, upon chemical modification of a HRP enzyme with adamantane groups, the enzyme could be immobilized at the surface maintaining a reasonable catalytic activity. This methodology allowed us to fabricate porous templates for catalytic reactions stable during several catalytic cycles. Moreover, the cyclodextrin porous films could be recovered by disruption of the host-guest interactions between the enzyme and the support. As a result, these versatile platforms could be employed for a large variety of catalytic reactions since the immobilization is based on the amino groups of the enzymes, the fabrication of multienzymatic systems or the preparation of enzymatic sensors among others.

Acknowledgements The authors gratefully acknowledge support from the Consejo Superior de Investigaciones Científicas (CSIC). Equally, this work was financially supported by the Ministerio de Economía y Competitividad (MINECO) through MAT201347902-C2-1-R, MAT2016-78437-R MAT2013-42957-R, MAT2015-65184-C22-R and CTQ2013-45538-P..

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We designed a microfluidic with porous reservoirs decorated with horseradish peroxidase (HRP). This enzyme, that binds to H2O2 forming a complex able to oxidize different compound, was reversible anchored thus permiting both the use of the support as catalyst as well as the recovery of the enzyme. 348x251mm (96 x 96 DPI)

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Microfluidic Reactors Based on Rechargeable Catalytic Porous

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