Polymer Chemistry c6py02086k

1

One-step preparation of surface modifiedelectrospun microfibers as suitable supportsfor protein immobilization

Guillaume Martrou, Marc Léonetti, Didier Gigmes andThomas Trimaille

Surface modified microfibers were prepared in a one-stepprocess, and were prone to retain the activity and improvethe stability of immobilized enzymes.

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Title: One-step preparation of surface modified electrospun microfibers as suitable supports for proteinimmobilization

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PolymerChemistry

PAPER

Cite this: DOI: 10.1039/c6py02086k

Received 2nd December 2016,Accepted 13th February 2017

DOI: 10.1039/c6py02086k

rsc.li/polymers

One-step preparation of surface modifiedelectrospun microfibers as suitable supportsfor protein immobilizationQ1 †

GuillaumeQ2 Martrou,a,b Marc Léonetti,b Didier Gigmesa and Thomas TrimailleQ3 a

We have here developed a straightforward one-step route to surface modified polystyrene (PS) based

microfibers for protein/enzyme immobilization. Our approach consists of wet electrospinning of a

poly(styrene-alt-maleic anhydride) (PSMA) polymer in an aqueous solution collector which contains the

(macro)molecules to be coupled, here PEG diamine (PEGDA) or hexamethylene diamine (hexDA). The

amino groups on the fiber surface were then exploited for immobilization of the horseradish peroxidase

(HRP) enzyme. The immobilized HRP amounts were higher on the PEG- and hexyl-modified fibers than

on the non-modified PSMA ones. The HRP catalytic activity was evaluated with 2,2’-azino-bis(3-ethyl-

benzothiazoline-6-sulfonic acid) (ABTS) as a substrate. While the retained activity of the enzyme immobi-

lized on unmodified PSMA microfibers was only 2.4% of free enzyme, that of the enzyme immobilized on

the PEGylated fibers increased to 34%. As a comparison, HRP fixed on hexyl-functionalized fibers exhi-

bited a retention activity of 19.7%, showing the impact of a PEG spacer on HRP activity. HRP immobili-

zation on PEG and hexyl-coated fibers had also a beneficial impact on enzyme storage stability. This study

highlights the impact of surface properties on the activity of the immobilized enzyme and provides a

convenient route to simultaneous elaboration/modification of fibers, for suitable protein fixation.

Introduction

Surface immobilization of proteins/enzymes is of fundamentalimportance in the area of bio-sensors, diagnostics, andcatalysis.1–3 It allows easy separation from solution productsand convenient reusability, greatly reducing process costs.Among the supports for protein fixation, electrospun micro/nanofibers have gained considerable interest over the lastdecade.4–6 They indeed present a large surface area, enablingincreased immobilized amounts and thus potential improve-ment of the sensitivity detection of (bio)molecules (immuno-assays)7 and/or enzyme catalyzed reaction efficiency.8–10

Compared to other nanomaterials such as nanoparticles orcarbon nanotubes, electrospun fibers are easier to be recoveredand reused since electrospinning can generate continuousnanofibers. In addition, the electrospinning process is versa-tile, highly reproducible and is now applicable to a wide rangeof (co)polymers.11,12

Immobilization often induces a loss of enzyme activity com-pared to the soluble free enzyme, due to the conformationalchanges occurring following immobilization.4,13–15 This can beinfluenced by many factors, including the enzyme itself, thelocation of the enzyme on or within the support, or themethod of immobilization and particularly the physical andchemical characteristics of the support.2 Despite intenseresearch on these aspects, many different (and sometimes con-tradictory) results on how enzymes are affected by immobili-zation are observed.16 Still, reports tend to show that hydro-phobic materials are particularly prone to induce proteinunfolding and further inactivation, at sufficient coverage.17,18

In comparison, hydrophilic ones, such as hydrogel-likematerials based on polyethylene glycol (PEG), have rather abeneficial impact on enzyme stability,19–22 which is parti-cularly attributed to reduced non-specific interactions andincreased mobility of the protein in a water enriched bio-friendly environment. Although PEG has shown great advancesfor proteins in the therapeutic field (protein “pegylation”), itsbeneficial impact on enzyme activity and stability has beenless studied and needs to be further explored.

Polystyrene (PS) is a famous example of a hydrophobicpolymer that has been widely used for protein/enzyme immo-bilization, due to its inexpensive and high workability,23–26

and a number of studies have been dedicated to advantageous†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6py02086k

aAix Marseille Univ, CNRS, ICR, Marseille, FrancebAix Marseille Univ, CNRS, Centrale Marseille, IRPHE, Marseille, France.

E-mail: thomas.trimaille@univ-amu.fr

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electrospun polystyrene fibers to this aim.27–30 PS hydrophobicsurfaces are however known to induce significant denaturationand inactivation of adsorbed enzymes.17,25,31 Therefore, thereare still challenges to develop versatile approaches for prepar-ing PS based fibers with tunable surface properties, prone toretain activities of the immobilized enzymes. To date, proteinattachment has been typically achieved onto fibers preparedfrom PS containing reactive maleic anhydride units, enablingcovalent coupling through anhydride–amine reaction.28 Thesurface maleic anhydride groups can also be used to post-functionalize the fibers with hydrophilic spacers such as PEG toimprove enzyme activity retention.32,33 However this approachrequires additional chemistry steps for fiber coating. Morerecently, An et al. reported post-functionalization of purePS nanofibers to generate surface amine groups for enzyme(alcohol dehydrogenase) immobilization through amidifica-tion in the presence of N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) coupling agent.34 Thisapproach provided acceptable enzyme stability, but still relieson a tedious two-step post-functionalization process.

In this work, we precisely developed a straightforward andversatile one-step electrospinning-based approach for the prepa-ration of PS based microfibers with tailored surface properties,such as improved hydrophilicity with amine groups and PEGspacing, and assessed their impact on the immobilization andactivity of the well-known horseradish peroxidase (HRP)enzyme. We particularly showed that these surface features werehighly beneficial for enzyme activity retention and stability.

ExperimentalMaterials

Poly(styrene-alt-maleic anhydride) (PSMA, Mn = 350 000 g mol−1,Fig. S1, ESI†), poly(ethylene glycol) bis(aminopropyl) terminated(PEGDA, Mn = 1500 g mol−1), hexamethylene diamine (hexDA),horseradish peroxidase (HRP, 44 kDa, pI = 7.2), N-(3-dimethyl-aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC),N-hydroxysuccinimide (NHS), 2,2′-azino-bis(3-ethylbenzothi-azoline-6-sulfonic acid) diammonium salt (ABTS) and hydrogenperoxide (30% in water) were purchased from Sigma. All sol-vents were of analytical grade and used as received. Phosphatebuffer saline (PBS) pH 7.4 was from Fisher Scientific.

One step preparation of surface modified fibers

PSMA dissolved in a 1 : 2 DMF : acetone solution (15 wt%) waselectrospun in a solution of PEGDA at 1 mg mL−1 or hexDA at0.077 mg mL−1 (0.67 µmol mL−1) in PBS buffer pH 7.4 used asthe collector. The working distance between the capillary andthe collector was 8.5 mm. A flow rate of 0.06 mL min−1 and avoltage of 3.2 kV were used. These conditions were necessaryto obtain a direct immersion of the electrospun solution in thecollector. For higher distance/smaller voltage the diameter/speed is too small to have enough momentum. For smaller dis-tance/higher voltage, the fiber can start burning. Referencenon-modified fibers were prepared in the same manner using

a pure PBS solution as the liquid collector. After 1 hour in thebuffer, the fibers were extensively washed with distilled water(5 times for 10 minutes) for removing the non-coupled aminocompounds, and dried under vacuum until constant weight.Fibers modified with PEGDA and hexDA were referred toas PSMA-PEG-NH2 and PSMA-hex-NH2, respectively. ControlPSMA fibers were also produced by conventional electro-spinning on a solid plan collector (aluminium), from the samesolvent mixture (acetone/DMF).30

The fibers were visualized by optical microscopy and scan-ning electron microscopy with a Xl30 ESEM FEI, coupled withEDAX Apollo 10. The fibers were characterized by ATR-FTIR(Spectrum Two, Perkin Elmer, equipped with a single reflec-tion diamond ATR accessory), and 1H NMR analysis (Bruker,400 MHz) in DMSO-d6. The molar amount of amines availableon the fiber surface was assessed by the fluorescamine assay35

as follows: fibers were dissolved in a mixture of DMSO : PBS2 : 1 at 2 mg mL−1. 90 µL of this solution were placed in a96-well microplate and 30 µL of fluorescamine solution(0.4 mg mL−1 in DMSO) was added. The reaction was allowedto proceed for 20 min in the dark, at room temperature, andthe fluorescence emission intensity was measured at 477 nmfor an excitation wavelength of 416 nm (TECAN apparatus). Itwas checked that PSMA fibers, analyzed under the same proce-dure, gave raise to negligible fluorescence. The calibrationcurve of fluorescence intensity (at 477 nm) versus amine con-centration obtained with PEGDA or hexDA allowed the deter-mination of the molar amount of amines per mg of fiber.

HRP immobilization on fibers

Immobilization of HRP on fibers (PSMA-PEG-NH2 or PSMA-hex-NH2) was performed by incubating 1 mL of a solution ofHRP at 0.5 mg mL−1 in PBS pH 7.4 (freshly prepared) with10 mg of fibers in a 2 mL Eppendorf tube. Then 10 µL of NHSwater solution (31 mg mL−1) and 10 µL of EDC water solution(50 mg mL−1) were added. The mixtures were gently stirred for4 hours. Control samples in the absence of EDC/NHS and HRPwere prepared as controls. The unmodified fibers of PSMAwere also used for HRP covalent immobilization. For thispurpose, the fibers of PSMA were first prepared by classicalelectrospinning on a plan plate collector (aluminium) to keepthe anhydride functions intact. After drying, 10 mg of PSMAfibers were incubated in 1 mL of a freshly prepared solutionof HRP at 0.5 mg mL−1 in PBS pH 7.4, for 4 hours. Thefibers were extensively washed with distilled water (5 times10 minutes) for removing the remaining free HRP and driedunder vacuum until constant weight.

HRP immobilized on the fibers was quantified by the BCAassay (Pierce kit). An accurate weight of the fiber-HRP sample(∼3 mg range, weighed with a Sartorius microbalance) wasfirst dissolved in 30 µL of DMF and 70 µL of water was furtheradded. Then 2 mL of BCA reactant was added. After incubationat 37 °C for 30 minutes, the absorbance was measured at562 nm. The absorbances were corrected from the residualabsorbance of the corresponding control fiber samples pre-pared in the absence of HRP. The amount of HRP was

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determined from a calibration curve established with solutionsof known HRP concentrations prepared under the same con-ditions. HRP desorption studies in sodium dodecyl sulfate(SDS) medium (5% in water) were performed on the fibers pre-pared with EDC/NHS to assess the covalent character of theimmobilization. Fibers prepared without EDC/NHS was used asthe control. In brief, a known amount of fibers was incubatedwith 200 µL of SDS solution for one hour. After centrifugation,the HRP present in the supernatant was quantified by BCA.

Enzyme activity

The activity of HRP was determined by using ABTS as the sub-strate at 25 °C. Typically, a precise predetermined mass offibers weighed with a Sartorius microbalance (0.5–0.9 mg, soas to be in the same HRP weight range, 6.5 µg) or 100 µL freeHRP (at 65 or 6.5 µg mL−1 in PBS, freshly prepared) wereplaced in a standard polystyrene UV-cuvette. Then 2 mL ofsolution consisting of 1.7 mmol L−1 ABTS and 0.83 mmol L−1

H2O2 in PBS pH 7.4 was added. The absorbance change of thesolution was measured every 15 s after rapid mixing, in aUV-vis spectrophotometer (Cary 50, VARIAN) at a wavelength of420 nm (molar extinction coefficient for the oxidation of ABTSat 420 nm: 36 000 M−1 cm−1) for 3 minutes. From the slopes ofthe kinetic curves, the specific activity (A) was expressed as theµmoles of consumed ABTS per minute and per mg of enzyme.The activity retention was defined as the ratio A(immobilizedenzyme)/A(free enzyme) × 100.

Stability experiments on immobilized or free HRP in PBSpH 7.4 at 4 °C were performed as follows: several samples of agiven type of fiber (0.5–0.9 mg range) were placed in aUV-cuvette and 1 mL of PBS was added. Free HRP samples inPBS (6.5 µg mL−1, 1 mL in a UV-cuvette) were also prepared.The samples were stored at 4 °C. At predetermined times, onesample was taken off and 2 mL of the above-mentioned PBSsolution of ABTS/H2O2 was added, for activity measurement,as described above. For the free HRP samples, 100 µL werewithdrawn and 900 µL PBS was added (10-fold dilution), fol-lowed by addition of 2 mL of ABTS/H2O2. The relative activitywas expressed as the specific activity (at time t ) to the initialspecific activity ratio, multiplied by 100.

For assessment of immobilized enzyme reusability, thefibers were placed in an empty UV cuvette. ABTS/H2O2 solution(2 mL) was added and the kinetics was measured as above.The solution was then removed, the fibers were rinsed withPBS, and 2 mL of fresh ABTS/H2O2 solution was added, andthe kinetics was again measured. This process was repeated8 times. The relative activity at each data point was calculatedfrom the ratio of activity for a given cycle to the initial activity,multiplied by 100.

Results and discussionOne step preparation of functionalized PS fibers by electrospinning

To date, electrospun fiber surface functionalization withappropriate macro- or bio-molecules is generally performed

after the electrospinning process, through chemical stepspossibly laborious and whose yield can be low due to reactionsoccurring in the heterogeneous phase.36 We here focused on astraightforward strategy to simultaneously form the fibers andfunctionalize them with appropriate polymers and/or reactivemoieties, based on wet electrospinning,37 which uses a liquidsolution as the collector. This technique has been reportedrecently, but only very few examples focused on the possibilityof fiber coating,38,39 and none were dedicated to the chemicalmodification of fibers. Our approach relied on electrospinningof a poly(styrene-alt-maleic anhydride) (PSMA) polymer in abuffered aqueous solution (PBS pH 7.4) used as the collectorand which contains the (macro)molecules to be coupled, herePEG diamine (PEGDA). As a result, the amino-molecules canspontaneously react with the anhydride moieties of theforming fibers in the aqueous medium. The process was pri-marily optimized using pure PBS (absence of PEGDA) as theliquid collector. Under appropriate conditions (see theExperimental section), we could produce microfibers able topenetrate the liquid and overcome the water surface tension,an important problem to solve. The process was then appliedusing a PEGDA solution in PBS pH 7.4 (1 mg mL−1) as thecollector liquid. PSMA fibers produced in pure PBS weresystematically used as a reference in the following for the sakeof comparison.

The fibers were visualized by optical microscopy and scan-ning electron microscopy (Fig. 1). The average diameter was7.0 µm (±1.5 µm) for the fibers prepared in the presence ofPEGDA, and 6.1 µm (±1.45 µm) for the reference fibers inpure buffer. Compared to the control fibers of PSMA classi-cally obtained by conventional electrospinning using a solidplan collector (Fig. S2, ESI†), the fibers obtained by the wetprocess had a higher diameter. It is here important to pointout the fiber swelling behavior induced by the aqueoussolvent used in the wet electrospinning process. This swellingis favored by the increased hydrophilic character of theinvolved polymers following penetration in the aqueousphase, for either the PEG modified PSMA or PSMA alone,whose anhydride functions were partly hydrolyzed intocarboxylate groups following immersion in the PBS buffer.The fibers also presented more pronounced entanglementsthan that observed by conventional electrospinning, aspreviously observed.37

Energy dispersive X-ray (EDX) combined with SEM was usedto assess the structural composition of the fibers (Table S1†).Sodium was present in 3 at% in reference PSMA fibers as acounter-ion of the carboxylate groups arising from hydrolysisof the anhydride functions in PBS pH 7.4. As a comparison,the PSMA fibers prepared in the presence of PEG showed alower sodium content (0.66 at%), as well as the presence ofnitrogen (2.05 at%) and an increased oxygen content (24 at%,against 19 at% for reference PSMA fibers), supporting success-ful fiber functionalization with PEG. The nitrogen content inthe PSMA reference sample (1.3 at%) was due to the residualDMF solvent, used in the electrospinning process, which wasevidenced by 1H NMR analysis (Fig. 2a).

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The PEG content in fibers was further quantified by1H NMR analysis of the fibers dissolved in DMSO-d6, compar-ing integral of the methylene proton peaks of the PEG chain at3.5 ppm with those of the peak of the aromatic protons of thePSMA at 6.5–7.5 ppm (Fig. 2). By varying the concentration ofPEGDA in the solution collector (0.05 to 1 mg mL−1), theamount of PEG fixed on the fibers could be modulated from33 to 145 µg mg−1 fiber (Fig. 3). The PSMA-PEG-NH2 fiberswere also characterized by ATR-FTIR analysis and compared tothe control PSMA fibers (Fig. 4). Apparition of the absorp-tion band at 1090 cm−1, characteristic of ether functions(CH2–O–CH2), evidenced the presence of PEG on the fibers. Anincreased intensity of this band was observed with the increas-ing concentration of the PEG collector solution, with a con-comitant decreasing intensity of the C–O band (1200 cm−1)relative to the anhydride functions (and carboxylic acidgroups), indicating the covalent nature of the coupling (for-mation of amide bond). Amide characteristic bands were notclearly observed on the spectra, and became more visible when

Fig. 1 Optical microscopy (A, B) and SEM (C–F) images of PSMAreference fibers obtained from PBS solution as a collector without PEG(A, C, E) and fibers obtained from PBS solution containing PEG at1 mg mL−1 (B, D, F).

Fig. 2 1H NMR analysis in DMSO-d6 of the PSMA-PEG fibers obtainedfrom the collector PEG solution at (a) 0 mg mL−1 (reference PSMAfibers), (b) 0.05 mg mL−1, (c) 0.1 mg mL−1, (d) 0.5 mg mL−1, (e) 1 mg mL−1;stars (*) indicate solvent peaks arising from DMSO-d6 solvent.

Fig. 3 PEG immobilized amounts as a function of PEG concentration ofthe collector solution (PBS, pH 7.4) used in the wet electrospinningprocess.

Fig. 4 ATR-FTIR analysis of PSMA-PEG-fibers obtained from the collectorPEG solution at (a) 0 mg mL−1 (reference PSMA fibers), (b) 0.05 mg mL−1,(c) 0.1 mg mL−1, (d) 0.5 mg mL−1, (e) 1 mg mL−1.

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subtracting the reference spectrum of PSMA fibers from thePEG-PSMA fibers (1660 cm−1 and 1595 cm−1, Fig. S3†). Finally,increasing –CH2– band intensity at 2850 cm−1 with PEG solu-tion concentration was again indicative of the increasing PEGcontent in the fibers.

Using the more concentrated PEG solution (1 mg mL−1),the amount of PEG on fibers was 145 µg mg−1 of fiber(i.e. 0.097 µmol mg−1). This high value could suggest that apart of PEG chains was buried in the fibers during formation.It is worth discussing here more in detail the process of fiberformation: due to a short distance between the capillary andthe solution surface (required for penetration in the solution),the PSMA polymer jet from the capillary is still largely swollenby the solvents (acetone and especially DMF, less volatile)when it penetrates the aqueous solvent. Upon penetration inaqueous solution, the final fibers are formed through solventdiffusion in water, and simultaneous coupling of the aminoPEG present in the medium occurs. In other words, couplingproceeds on the still swollen forming fibers from which thesolvent is being excluded. This coupling can thus take place,to a certain extent, in the inner part of the forming fibers andnot only on the surface. In that sense, the functionalizationprocess has to be considered different from classical surfacecoupling onto the already prepared fibers, and most probablyexplains the high PEG fiber content achievable, as well as theincreased diameter of the fibers compared to PSMA fibers pre-pared in the absence of PEG. Moreover, the hydrophilizationof the fibers upon hydrolysis and/or amine coupling (resultingin the formation of carboxylate groups that can repulse eachother) contributes to favor PEG functionalization.

These PEG-functionalized fibers were used for furtherstudies. Fibers modified with a hexDA analog were also pre-pared through this process, at the same molar concentrationas that of 1 mg mL−1 PEG solution, namely 0.077 mg mL−1

(0.67 µmol mL−1) of hexDA in PBS. The fibers had a diameterof 7.75 ± 1.24 µm (Fig. S4†) and EDX analysis supported suc-cessful functionalization (Table S1†).

The presence of amines at the fiber surface was attested bythe fluorescamine method as described in the Experimentalsection. The amine surface density was found to be about0.065 ± 0.004 µmol mg−1 of PEG functionalized fibers,and very similar for the hexyl functionalized ones (0.063 ±0.003 µmol mg−1). Based on the PEG coated amounts of0.097 µmol mg−1 (and considering that PEG bears two aminefunctions), one could expect an amine density of 0.097 µmol mg−1

if PEG was exclusively coupled by one of its amines. Thisfurther consumption of amines suggests a partial crosslinkingof the fibers. This was also visually observed by a hard solubil-ization of the functionalized fibers in DMSO compared to thereference PSMA fibers prepared in the absence of a diaminecompound.

Enzyme immobilization on the surface modified fibers

The HRP enzyme was immobilized on the PSMA-hex-NH2 andPSMA-PEG-NH2 functionalized fibers through the EDC/NHSstrategy in PBS buffer pH 7.4 (Fig. 5B and C). As a reference,

HRP was also immobilized onto the unmodified PSMA fibersunder the same conditions (Fig. 5A). Here, the immobilizationproceeded through reaction of the lysine and N-terminalamines of HRP with the anhydride functions of the PSMAfibers previously prepared on the plan solid collector (samplereferred to as PSMA-HRP).

The HRP immobilized amounts were determined by theBCA assay. As shown in Table 1, a higher density of HRP wasobtained for the hexyl-NH2 and PEG-NH2 modified fibers thanfor the unmodified ones. This could be primarily ascribed tothe different route of immobilization. Indeed, the fixation onthe PSMA reference fibers involved the amine groups of theenzyme (6 Lys residues) whereas that on the hexyl- and PEG-modified fibers involved its carboxylic functions, much morepredominant in the molecule (28 Asp/Glu residues).40 Thishigher HRP density on the functionalized fibers could also bereasonably attributed to the improved accessibility of the func-tions at the fiber surface (through hexyl/PEG spacing) for inter-actions with the protein.

Despite the same route of immobilization for the hex-modi-fied and the PEG-modified fibers, and similar amine density,the amount of HRP adsorbed was slightly lower for the PEGmodified fibers. This could be due the protein repellent effectof PEG, even if the PEG spacer could also be supposed to makethe amine functions more accessible for reaction with HRP.

As a reference, immobilization experiments were performedon the hexyl- and PEG-fibers under the same conditions (PBSpH 7.4) but in the absence of EDC/NHS. HRP immobilizationwas far from negligible (11.8 and 10.8 µg mg−1 of fibers,respectively) though lower than that obtained for EDC/NHS

Fig. 5 Schematic view of the different HRP functionalized fibers:PSMA-HRP (A), PSMA-hex-HRP (B), and PSMA-PEG-HRP (C).

Table 1 HRP immobilized amounts on unmodified and modified fibersand activity retention, in PBS pH 7.4

FibersImmobilized HRP(µg mg−1 fibers)

Activityretention (%)

PSMA-HRP 7.6 ± 0.4 2.4 ± 0.8PSMA-hex-HRP 13.4 ± 0.5 19.7 ± 4.1PSMA-PEG-HRP 12.6 ± 0.3 34.0 ± 5.0

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coupling, and due to electrostatic interactions between theslightly anionic HRP (pI = 7.2) and protonated amines of hex/PEG. These results were consistent with earlier studies report-ing electrostatically driven adsorption of HRP onto poly(4-vinylpyridine) polycations41,42 at the same pH (PBS). Furthermore,despite the well-known protein repellent effect of PEG, recentstudies have shown that proteins, such as HRP43 and pinekernel peroxidase44 could be non-covalently immobilized ontomaterials coated with PEG of sufficiently low molecularweight.

Considering these non-negligible amounts of adsorbedHRP, it was necessary to prove that HRP was well covalentlyattached on the fibers in the presence of EDC/NHS. For thispurpose, we have further performed enzyme desorptionstudies with different fibers in the presence of sodium dodecylsulfate (SDS) as a desorbing agent, which is well known forpeptides/proteins to disrupt non-covalent bonds. The enzymereleased was then quantified by BCA in the supernatant(Fig. S5†). Interestingly, in the presence of SDS, negligible de-sorption was observed for the HRP immobilized on PEG- andhexyl-functionalized fibers in the presence of EDC/NHS, whilethe HRP passively immobilized on these fibers in the absenceof the coupling agents was largely desorbed. This unambigu-ously evidences that the HRP immobilized in the presence ofEDC/NHS is quasi entirely attached on the fibers in thecovalent manner. The fibers with passively adsorbed HRP werenot further investigated regarding HRP activity, since they aremore prone to desorption than a covalently attached enzyme.

Enzyme activity

The activity of the enzyme, free or covalently immobilized onthe various fibers, was assessed with ABTS as the substrate inthe presence of hydrogen peroxide. The kinetics of the reactionwas followed by measuring the absorbance of the forming oxi-dized ABTS at 420 nm as a function of time, allowing the deter-mination of the initial rate (slope) and further specific activityas well as retention activity compared to free HRP. The weightof different fibers was adjusted so that the HRP mass was inthe same range (∼6.5 µg). Running the experiment with thismass of free HRP, the kinetics of the oxidation was a bit hardto monitor due to saturating absorbance values at early times.The experiment was then also typically run with a ten-foldlower mass, namely 0.65 µg of free HRP.

As shown in Table 1, direct immobilization of HRP on theunmodified PSMA fibers (PSMA-HRP) led to low activity reten-tion (2.4%), similarly as previously reported for the acetyl-cholinesterase enzyme immobilized in the same manner ontoPSMA fibers.32 This was most probably due to importantprotein conformational changes and further inactivationoccurring upon immobilization through the surface anhydridefunctions of the fibers. Much higher activity retention wasobserved for the enzyme immobilized on the hexyl modifiedfibers (19.7% activity retention). Here, the spacing effect and,to a less extent, higher hydrophilicity brought by the hexyl-amine moiety presumably induced a more favorable confor-mation of the immobilized enzyme, i.e. a better accessibility of

the enzyme active sites for the substrate. The activity retentionwas further significantly improved for the HRP immobilizedon the PEG functionalized fibers (34%), clearly highlightingthe beneficial impact of the hydrophilic PEG spacer, byimproving again the flexibility/mobility of the protein in thewater enriched environment.

Storage stability in PBS pH 7.4 at 4 °C of the immobilizedHRP was evaluated and compared to that of the free enzyme.As shown in Fig. 6, the activity of the free enzyme decreasedsignificantly over 11 days, contrary to the activity of theimmobilized enzymes. While the activity of the HRP immobi-lized on hexyl functionalized fibers remained nearlyunchanged over one week, the activity of the enzyme of PEGmodified fibers even tended to slightly increase. This surpris-ing result may be attributed to the swelling process takingplace for these hydrophilized PEG functionalized fibers withstorage time in the buffer, presumably making the enzymemore accessible to the substrate. The reusability of HRPimmobilized on the PEG- and hexyl-functionalized fibers wasfinally evaluated over 10 cycles, in PBS pH 7.4 (Fig. S6†).Activity decreased over repeated cycles for both fibers, but HRPimmobilized on the PEG fibers showed slightly improved per-formances compared to the hexyl ones.

Taken together, these results clearly show the highly ben-eficial impact of the PEG environment on enzyme activity pres-ervation, as well as storage and operational stability.

Conclusions

We have here developed a straightforward one-step route tosurface modified polystyrene (PS) based microfibers based onthe wet electrospinning of PSMA, using a buffered liquid col-lector containing the molecules of interest (PEGDA, hexDA)that can be spontaneously coupled on the forming fibers. Thelatter could be used for facile and efficient immobilization ofthe HRP enzyme. Spacer groups, particularly PEG, induced ahigher enzyme activity compared to direct PSMA fibers andimproved stability over that of the free enzyme. This study

Fig. 6 Storage stability (PBS pH 7.4, 4 °C) of HRP immobilized on hexyl(▲) and PEG (●) functionalized fibers, compared to free HRP (○), bymeans of relative activity (% of initial activity).

Paper Polymer Chemistry

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highlights the impact of surface properties on the activity ofan immobilized enzyme, and provides a novel straightforwardand relevant approach to simultaneously elaborate/modifyfibers potentially made from other reactive polymers of inter-est, such as those based on N-succinimidyl esters, for furtherprotein/enzyme immobilization.

Acknowledgements

The authors acknowledge Carnot STAR Institute and PACAregion for financial support, and A. Tonetto from Aix-MarseilleUniversity (PRATIM) for EDX-SEM experiments.

Notes and references

1 J. Goddard and J. Hotchkiss, Prog. Polym. Sci., 2007, 32,698–725.

2 W. Tischer and F. Wedekind, in Biocatalysis – FromDiscovery to Application, ed. W.-D. Fessner, A. Archelas,D. C. Demirjian, R. Furstoss, H. Griengl, K.-E. Jaeger,E. Morís-Varas, R. Öhrlein, M. T. Reetz, J.-L. Reymond, M.Schmidt, S. Servi, P. C. Shah, W. Tischer and F. Wedekind,Springer Berlin Heidelberg, Berlin, Heidelberg, 1999,pp. 95–126.

3 L. Allard and T. Trimaille, WO 2016/027021, 2016Q4 .4 Z.-G. Wang, L.-S. Wan, Z.-M. Liu, X.-J. Huang and Z.-K. Xu,

J. Mol. Catal. B: Enzym., 2009, 56, 189–195.5 D. N. Tran and K. J. Balkus, Top. Catal., 2012, 55, 1057–

1069.6 N. Keloglu, B. Verrier, T. Trimaille and J. Sohier, Colloids

Surf., B, 2016, 140, 142–149.7 S. Chantasirichot and K. Ishihara, Biosens. Bioelectron.,

2012, 38, 209–214.8 J. Xie and Y.-L. Hsieh, J. Mater. Sci., 2003, 38, 2125–

2133.9 J. Niu, J. Xu, Y. Dai, J. Xu, H. Guo, K. Sun and R. Liu,

J. Hazard. Mater., 2013, 246–247, 119–125.10 R. Xu, C. Chi, F. Li and B. Zhang, Bioresour. Technol., 2013,

149, 111–116.11 T. J. Sill and H. A. von Recum, Biomaterials, 2008, 29, 1989–

2006.12 D. Li and Y. Xia, Adv. Mater., 2004, 16, 1151–1170.13 T. Zhang, X.-L. Xu, Y.-N. Jin, J. Wu and Z.-K. Xu, J. Mol.

Catal. B: Enzym., 2014, 106, 56–62.14 A. M. Klibanov, Anal. Biochem., 1979, 93, 1–25.15 S. M. Lane, Z. Kuang, J. Yom, S. Arifuzzaman, J. Genzer,

B. Farmer, R. Naik and R. A. Vaia, Biomacromolecules, 2011,12, 1822–1830.

16 L. Cao, in Carrier-bound Immobilized Enzymes, Wiley-VCHVerlag GmbH & Co. KGaA, 2006, pp. 1–52.

17 S. Di Risio and N. Yan, Colloids Surf., B, 2010, 79, 397–402.18 S. Sharma, B. J. Berne and S. K. Kumar, Biophys. J., 99,

1157–1165Q5 .19 S. Zhang, Nat. Mater., 2004, 3, 7–8.

20 S. Mastour Tehrani, Y. Lu, G. Guerin, M. Soleimani,D. Pichugin and M. A. Winnik, Biomacromolecules, 2015,16, 3134–3144.

21 A. M. Mariani, M. E. Natoli and P. Kofinas, Biotechnol.Bioeng., 2013, 110, 2994–3002.

22 Y. Wang and Y.-L. Hsieh, J. Polym. Sci., Part A: Polym.Chem., 2004, 42, 4289–4299.

23 A. A. Ansari, N. S. Hattikudur, S. R. Joshi andM. A. Medeira, J. Immunol. Methods, 1985, 84, 117–124.

24 A. Niveleau, C. Bruno, E. Drouet, R. Brebant, A. Sergeantand F. Troalen, J. Immunol. Methods, 1995, 182, 227–234.

25 K. Yanagisawa, T. N. Murakami, Y. Tokuoka, A. Ochiai,M. Takahashi and N. Kawashima, Colloids Surf., B, 2006,48, 67–71.

26 Y. Zhang, L. Li, C. Yu and T. Hei, Anal. Bioanal. Chem.,2011, 401, 2311–2317.

27 J. H. Kim, E. T. Hwang, K. Kang, R. Tatavarty and M. B. Gu,J. Mater. Chem., 2011, 21, 19203–19206.

28 S. Nair, J. Kim, B. Crawford and S. H. Kim,Biomacromolecules, 2007, 8, 1266–1270.

29 S. J. Lee, R. Tatavarty and M. B. Gu, Biosens. Bioelectron.,2012, 38, 302–307.

30 W. J. Cloete, C. Adriaanse, P. Swart and B. Klumperman,Polym. Chem., 2011, 2, 1479–1481.

31 J. E. Butler, L. Ni, W. R. Brown, K. S. Joshi, J. Chang,B. Rosenberg and E. W. Voss, Mol. Immunol., 1993, 30,1165–1175.

32 O. Stoilova, M. Ignatova, N. Manolova, T. Godjevargova,D. G. Mita and I. Rashkov, Eur. Polym. J., 2010, 46, 1966–1974.

33 L. Zhao, Q. Liu, S. Yan, Z. Chen, J. Chen and X. Li,J. Biotechnol., 2013, 168, 46–54.

34 H. An, B. Jin and S. Dai, Enzyme Microb. Technol., 2015, 68,15–22.

35 L. Allard, V. Cheynet, G. Oriol, G. Gervasi, E. Imbert-Laurenceau, B. Mandrand, T. Delair and F. Mallet,Bioconjugate Chem., 2004, 15, 458–466.

36 H. S. Yoo, T. G. Kim and T. G. Park, Nanofibers Regener.Med. Drug Delivery, 2009, 61, 1033–1042.

37 Y. Yokoyama, S. Hattori, C. Yoshikawa, Y. Yasuda, H. Koyama,T. Takato and H. Kobayashi,Mater. Lett., 2009, 63, 754–756.

38 X. Wang, M. Min, Z. Liu, Y. Yang, Z. Zhou, M. Zhu, Y. Chenand B. S. Hsiao, J. Membr. Sci., 2011, 379, 191–199.

39 Y. Zheng, J. Miao, N. Maeda, D. Frey, R. J. Linhardt andT. J. Simmons, J. Mater. Chem. A, 2014, 2, 15029–15034.

40 K. G. Welinder, Eur. J. Biochem., 1979, 96, 483–502.41 C. Sun, W. Li, Y. Sun, X. Zhang and J. Shen, Electrochim.

Acta, 1999, 44, 3401–3407.42 W. Li, Z. Wang, C. Sun, M. Xian and M. Zhao, Anal. Chim.

Acta, 2000, 418, 225–232.43 M. B. Fritzen-Garcia, F. F. Monteiro, T. Cristofolini,

J. J. S. Acuña, B. G. Zanetti-Ramos, I. R. W. Z. Oliveira,V. Soldi, A. A. Pasa and T. B. Creczynski-Pasa, Sens.Actuators, B, 2013, 182, 264–272.

44 M. B. Fritzen-Garcia, I. R. W. Z. Oliveira, B. G. Zanetti-Ramos, O. Fatibello-Filho, V. Soldi, A. A. Pasa andT. B. Creczynski-Pasa, Sens. Actuators, B, 2009, 139, 570–575.

Polymer Chemistry Paper

This journal is © The Royal Society of Chemistry 2017 Polym. Chem., 2017, 00, 1–7 | 7

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