Switchable Bioelectrocatalysis Controlled by Dual Stimuli-Responsive

Subscriber access provided by NEW YORK MED COLL

Article

Switchable Bioelectrocatalysis Controlled by Dual Stimuli-Responsive Polymeric Interface Onur Parlak, Md. Ashaduzzaman, Suresh B. Kollipara, Ashutosh Tiwari, and Anthony P. F. Turner ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06048 • Publication Date (Web): 06 Oct 2015 Downloaded from http://pubs.acs.org on October 8, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Switchable Bioelectrocatalysis Controlled by Dual StimuliResponsive Polymeric Interface Onur Parlak,1,† Md. Ashaduzzaman,1,2,† Suresh B. Kollipara,1 Ashutosh Tiwari1,3* and Anthony P. F. Turner1 1

Biosensors and Bioelectronics Centre, IFM, Linköping University, S-58183 Linköping,

Sweden 2

Department of Applied Chemistry and Chemical Engineering, University of Dhaka, Dhaka

1000, Bangladesh 3

†

Tekidag AB, UCS, Mjärdevi Science Park, Teknikringen 4A, SE 583 30 Linköping, Sweden

The authors contributed equally.

*Corresponding author. E-mail: [email protected], Tel: (+46) 13-28-2395 and Fax: (+46) 13-13-7568.

ABSTRACT: The engineering of bio-nano interfaces using stimuli-responsive polymers offers a new dimension in the design of novel bioelectronic interfaces. The integration of electrode surfaces with stimuli-responsive molecular cues provides a direct control and ability to switch and tune physical and chemical properties of bioelectronic interfaces in various bio-devices. Here, we report a dual-responsive bio-interface employing a positively responding dual switchable polymer, poly(NIPAAm-co-DEAEMA)-b-HEAAm to control and regulate enzyme-based bioelectrocatalysis. The design interface exhibits reversible activation-deactivation of bio-electrocatalytic reactions in response to change in temperature and to pH, which allows manipulation of biomolecular interactions to produce on/off switchable conditions. Using electrochemical measurements, we demonstrate that interfacial bio-electrochemical properties can be tuned over a modest range of temperature (i.e., 20-60 °C) and pH (i.e., pH 4-8) of the medium. The resulting dual-switchable interface may have 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

important implications for the design not only of responsive bio-catalysis and on-demand operation of biosensors, but also as an aid to elucidating electron-transport pathways and mechanisms in living organisms by mimicking the dynamic properties of complex biological environments and processes. KEYWORDS: Stimuli-responsive polymers, tunable biocatalysis, switchable bioelectronics, biointerfaces, tri-arm polymers, ATRP. BRIEFS: A dual-responsive polymeric bio-interface is capable of responding to change in temperature and pH, which can regulate and trigger bioelectrocatalytic systems. 1. INTRODUCTION Switchable bioelectronic interfaces, comprising stimuli-responsive materials integrated with electrodes, which have a distinct capability to alter their macroscopic properties on-demand are key elements for the construction of engineered enzyme-based bio-catalysis.1-4 Surfaces modified by suitable (macro) molecular units, which have the ability to mimic aspects of the natural environment of redox enzymes in terms of structure and functionality, 5 offer new opportunities to control their physical and chemical properties or to modulate the conversion of chemical and biochemical signals into electrical impulses, and vice versa. 6-9 The control of biocatalysis by real-life physical and chemical stimuli is vitally important to understand electron transport pathways in biochemical systems and the modulation of electron-transfer in redox biomolecules involved in physiological processes and therapy.10 Over the past decade, a variety of approaches have been developed to create various switchable bioelectronic interfaces, which were capable of changing their properties in response to several different stimuli. For instance, various signal-responsive materials -can positively respond to light irradiation, changes in temperature or pH, and electric or magnetic fields. Examples include polymers nanoparticles, (supra) molecules and self-assembled 2


Page 2 of 28

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


monolayers (SAM) which can be employed or modified on various nanostructured materials or electrode surfaces to produce switchable interfaces to control and modulate enzyme catalysis or the sensing of an analyte in response to external stimuli.5-9 Among the various responsive materials, polymers have multiple advantages over their counterparts due to their biocompatibility, fast response time, film-forming ability, easy of chemical modification and the possibility to combine various stimuli in one unit. 10-12 The several key aspects have recently attracted considerable interest in stimuliresponsive polymeric bio-interfaces. Switchable and/or tunable adhesion between stimuliresponsive polymers and proteins or cells has been investigated in order to obtain controllable adhesion, tissue engineering or bioseparation.13-15 A second key advance has been the ability to control and regulate exposure and masking of functional moieties at the biointerface, which is crucial for adjusting regulatory signals and modulation of biomolecular activity. 16 Moreover, the control of the permeability of chemicals through a porous membrane or the reversible interaction of biomolecules and ions with a surface, offers a unique opportunities for biosensing and biocatalysis.17-19 In this respect, surfaces combined with stimuliresponsive polymers emerge as a reasonable alternative to control many different physical and chemical features in many biological systems for various applications. 20 Controlled conformational changes of stimuli-responsive polymeric interfaces allow reversible changes of the interfacial properties under diverse external stimuli for switchable bioelectrocatalysis.21-26 Interactions at such interfaces have been controlled by various external stimuli, including changes in pH, electrochemical potential, temperature, magnetic field and light.27-36 However, using stimuli responsive polymeric interfaces that respond to only a single stimulus is insufficient to mimic complex biological environments.

There is an

obvious need to upgrade already existing methods and conditions by combining more than one environmental stimuli in a single polymeric unit. The incorporation of di- and/or multi3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

stimuli responsive interfaces can likely better adapt to and mimic biological environments and processes.37 It is believe that the evolution of recently emerging multi-stimuli responsive bio-interfaces will greatly enhance the versatility of materials and also provide more satisfactory expansions of electron-transport pathways and electron-transfer processes of redox-active biomolecules.1,

20

The present manuscript aims to address the design and development of a dual stimuli responsive

tri-arm block

co-polymer,

poly(NIPAAm-co-DEAEMA)-b-HEAAm that is

capable of positively responding to both temperature and pH change. The dual responsive property of the block

co-polymer is attributed

isopropylacrylamide-PNIPAAM) -

and

pH-sensitive

to

the thermoresponsive (poly-N-

(poly-N,N-diethylaminoethylmethacrylate

PDEAEMA) units in each polymer chain, whereas a non-responsive hydrophilic block

(poly-N-hydroxyethylacrylamide-PHEAAm) contributes solvated corona in aqueous solution. The whole block co-polymer exhibits reversible temperature-responsive phase transition in aqueous solution at the lower critical solution temperature (LCST) of 32 °C, due to having the PNIPAAM block in the polymer chain. In addition, presence of PDEAEMA blocks in the polymer chain brings the pH-sensitive character, which exhibits a reversible structural change upon a pH shift in solution. To the best of our knowledge, there has been no previous reports which combine a poly(NIPAAm-co-DEAEMA)-b-HEAAm tri-arm block co-polymer in bioelectrocatalytic processes. In this study,

the bio-interface consists of tri-arm block copolymer having

poly(NIPAAm-co-DEAEMA)-b-HEAAm and

glucose oxidase (GOx, from Aspergillus

niger), which are rationally assembled together on an electrode surface. At a desirably low temperature (i.e. 20 °C) and pH (i.e. pH 4) the interaction between NIPAAm and DEAEMA monomers was subverted and the block co-polymer became hydrophilic, thus the biosubstrate could freely access the enzyme facilitating bioelectrocatalysis. In contrast, at a 4


Page 4 of 28

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


relatively high temperature (i.e., 40 °C) and pH (i.e., pH 8) the coalescence between the functional groups in the polymer chain increased and the overall structure turned hydrophobic, which restricted the interaction between the enzyme and its substrate. This provides the first example of responsive bioelectrocatalysis being achieved using a tri-arm block co-polymer by controlling the external temperature and pH in an on/off switchable model. The on/off switching behaviour of the dual-responsive polymer-modified electrode was demonstrated using different electroactive redox couples having different charges including negatively charged ferro/ferri cyanide Fe[(CN)6 ]3-/4- and ferrocene carboxylic acid (FeCOOH), and positively charged ruthenium (III) hexamine trichloride, and neutral hydroquinone as model electroactive probes for electrochemical measurements. 2. EXPERIMENTAL DETAILS Synthesis

of

the

tri-arm

initiator

Anhydrous

1,3,5-trihydroxybenzene

1,3,5-(2ʹ-bromo-2-methyl-propionato)benzene:

(11.90

mmol)

was

dissolved

in

anhydrous

tetrahydrofuran (THF, 120 mL) under a nitrogen gas environment, and triethylamine (TEA, 30 mL) was added as a catalyst for the reaction in a dried round-bottom flask. A THF (80 ml) solution with 2´-bromo-2-methylpropionyl bromide (110.9 mmol) was slowly added into the reaction flask in an ice-bath. Following this addition, a turbid solution appeared and the solution in the flask was stirred for an additional 24 h at room temperature (RT) under continuous nitrogen flow. The by-product salt (triethylamine/hydrobromic acid) and excess TEA were removed from the THF solution by filtration and extraction with a NaHCO 3 aqueous solution (2% w/v). The solution was then instantly dewatered by the addition of anhydrous Na2 SO4 ; the tri-functional initiator, 1,3,5-(2ʹ-bromo-2-methyl-propionato) benzene was obtained in a 73 mol% yield as a brown crystalline product by evaporation. The purity was checked by elemental analysis (theoretical: H% 3.68, C% 37.68, experimental: H% 3.86, 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

C% 37.87) and 1 H-NMR in CDCl3 after vacuum drying. 1 H-NMR revealed that two singlet signals for the methyl (-CH3 ) protons and the phenyl (C 6 H3 ) protons were present from the chemical shifts, δ = 1.90-2.12 ppm (18H, s) and δ = 6.91-7.00 ppm (3H, s), respectively (Figure S2). The area signal ratio of the phenyl and methyl protons was 1:5.98, which corresponded with the theoretical ratio of 1:6. Synthesis of smart macroinitiator: A dimethylsulfoxide solution (1 mL) of the trifunctional initiator, 1,3,5-(2ʹ-bromo- 2-methyl-propionato) benzene (0.25 mmol) and Nisopropylacrylamide (NIPAAm) and diethylaminoethylmethacrylate (DEAEMA) monomers (50 mmol, each) were added into isopropanol (10 mL) in a round-bottom flask and purged with argon. Stirring was carried out with a magnetic stirrer and the solution was purged continuously. 1, 1’, 4, 7, 10, 10ʹ-hexamethyl triethylene tetramine (HMTETA, 0.3 mmol) and Cu(I)Br (0.25 mmol) were then successively added into the flask at 60 °C. After 24 h, the polymerisation was terminated, air was allowed to enter the reactor and a highly viscous green polymer solution was obtained. Purification was conducted by dialysing entirely against deionised water using a dialysis bag (3,500 Mw) at room temperature (RT). The product was dried in a freeze-dryer. Fabrication of dual stimuli-responsive polymer: Tri-arm poly(NIPAAmn-co-DEAEMABr)3 (0.5 g, 0.013 mmol) and HEAAm monomer (5.3 g, 46.10 mmol) was dissolved in an ethanol/water mixed solution (v/v 10:1, 11 mL), and this solution was deoxygenated with argon. HMTETA (0.15 mmol) and Cu(I)Br (0.12 mmol) were successively added to the flask and the reaction was continued at RT for 24 h. A light-green highly viscous solution was thus obtained by warming the flask, indicating the immediate initiation of an exothermic block formation reaction. The clear, transparent, homogeneous, jelly-like polymer product was purified by dialysing against deionised water. 6


Page 6 of 28

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fabrication of switchable electrode interface: Prior to coating, glass carbon electrodes (GCEs) were carefully polished using 1.0, 0.3, and 0.05 micron Buehler alumina slurry on a Buehler polishing micro-cloth (Buehler, Ltd. USA) and washed with deionised water. A mixture of 20 μL aqueous solutions of tri-arm poly(NIPAAm-co-DEAEMA)-b-HEAAm polymer (10 mg/mL) and 10 μL of glucose oxidase (GOx, 10 mg/mL) in 10 mM PBS was then drop-casted onto the electrode surface, following which the electrode was kept in the cold room at 4 o C overnight. Temperature-controlled scanning

electron microscopy experiments: The structural

change of tri-arm poly(NIPAAm-co-DEAEMA)-b-HEAAm coated electrode surface was investigated by in-situ temperature controlled scanning electron microscopy technique (insitu SEM). The temperature of the surrounding was controlled using additional electrical heating/water cooling attachement, as shown in Figure S6 (Supporting Information). The pH control is provided by freezing modified electrodes in liquid nitrogen before measurements. 3. RESULTS AND DISCUSSION Scheme 1 shows the reaction steps for the synthesis of the dual switchable tri-arm block copolymer

poly(NIPAAm-co-DEAEMA)-b-HEAAm

via

atom

transfer

radical

polymerisation (ATRP). The tri-arm pH and thermo-sensitive polymer consists of inner pH and thermo-sensitive blocks and an outer hydrophilic poly(HEAAm) segment. The dual stimuli polymer dissolved in water depending on pH and/or temperature; above the LCST and critical pH there was a phase transition of DEAEMA/NIPAAm units into a homogeneous dispersion. In first a step, 1,3,5-(2´-bromo-2-methyl propionato) benzene, as a trifunctional initiator, was synthesised by esterification from 1,3,5-trihydroxybenzene and 2′-bromo-2methyl prepared

propionyl by

macroinitiator.

bromide.

ATRP

Secondly,

poly(NIPAAm-co-DEAEMA)-b-HEAAm

of HEAAm using

tri-arm poly(NIPAAm-b-DEAEMA)3

was as a

ATRP with copolymerisation of NIPAAm and DEAEMA and block 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 28

formation with HEAAm were conducted in a mixture of dimethylsulfoxide (DMSO) and isopropanol at 60 °C, and in aqueous ethanol (ethanol: water = 10:1 v/v) at RT to produce copolymer and block copolymer, respectively. In both ATRP reactions, 1,1′,4,7,10,10′hexamethyl triethylene tetramine (HMTETA) and Cu(I) Br were used as ligand and catalyst, respectively. The block co-polymers synthesised by atom transfer radical polymerisation were characterised by attenuated total reflectance-infra red spectroscopy (ATR-IR) and the results are shown in Figure S1. The characteristic peaks of amide I (C=O stretching) and amide II (N–H bending) at approximately 1630 and 1549 cm-1 were attributed to the amide bond (―CO―NH―) of the NIPAAm units. Whereas, the peak at 1740 cm-1 was accounted for the carbonyl bond (=C=O) of DEAEMA units of the core segment (macroinitiator). The distinctive ―OH peaks of HEAAm were observed at approximately 1062 cm-1 (C–O–H stretch) and 3300 cm-1 (O–H stretch). The enlargement of the amide bond and introduction of a hydroxyl (–OH) bond indicates the formation of a HEAAm segment in the block copolymer structure. Additionally, Figure S2 demonstrates the degree of polymerisation of NIPAAm, DEAEMA and HEAAm units estimated from 1 H-NMR spectral analysis of the initiator,

tri-arm

poly(NIPAAm15 -co-DEAEMA60 )

and

tri-arm

poly((NIPAAm15 -co-

DEAEMA60 )-b-HEAAm80 . The overall Mn value of the block copolymer was calculated to be 66,600 g mol-1 . Table S1 in the Supporting Information shows a peak area at δ = 6.8-7.2 for phenyl protons of initiator, which was considered as a reference peak value for estimation of NIPAAm and DEAEMA units. Subsequently, the area of isopropyl =CH― (δ = 3.8-4.0) was further considered to measure the number of HEAAm units. The driving force of aggregation was the hydrophobicity of NIPAAm and DEAEMA units

in

different

environments.

The

dispersion

ability

8


of

the

tri-arm copolymer

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(macroinitiator) and block copolymer was studied. The results show that the size of assynthesised macro initiator and tri-arm block co-polymers can be tuned by temperature and pH. Under temperature conditions below the LCST and pH 7.0, the overall structure moves to macroscopic aggregation by dehydration of the NIPAAM and DEAEMA units. The aqueous solution of block copolymer was transparent in the temperature region from 20 to above 60 °C. The solution did not show any obvious change at concentrations up to 3.0×10 -6 M (0.2 mg/mL), indicating that the increased hydrophilicity of outer HEAAm chains facilitate the dispersibility of the block copolymer. The dispersion properties of the block copolymer at lower concentration were investigated using dynamic light scattering. We selected 0.1 mg/mL (1.5×10 -6 M) as a moderate concentration of polymer in aqueous solution, because 10 -8 M was too low to monitor by dynamic light scattering (DLS) at pH 4.5, and macro aggregation was observed above 10-6 M even below the LCST. Below the LCST and at very low particle concentration (1.5×10 -6 M), the size could not be measured by DLS. When the temperature was increased very slowly (typically

Switchable Bioelectrocatalysis Controlled by Dual Stimuli-Responsive

Recommend Documents