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Bioinspired design of a polymer-based biohybrid sensor interface
Sensors and Actuators B 251 (2017) 674–682
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Bioinspired design of a polymer-based biohybrid sensor interface Erdo˘gan Özgür a,b , Onur Parlak b , Valerio Beni b,c , Anthony P.F. Turner b , Lokman Uzun a,b,∗ a
Hacettepe University, Department of Chemistry, Ankara, Turkey Linköping University, Biosensors and Bioelectronics Centre, IFM, Linköping, Sweden c RISE Acreo, Research Institute of Sweden, Norrköping, Sweden b
a r t i c l e
i n f o
Article history: Received 24 January 2017 Received in revised form 23 April 2017 Accepted 8 May 2017 Available online 10 May 2017 Keywords: Biomimicry Amino acid Macroporosity Polymeric film Supramolecular self-assembly
a b s t r a c t The key step in the construction of efficient and selective analytical separations or sensors is the design of the recognition interface. Biomimicry of the recognition features typically found in biological molecules, using amino acids, peptides and nucleic acids, provides plausible opportunities to integrate biological molecules or their active sites into a synthetic polymeric backbone. Given the basic role of functional amino acids in biorecognition, we focused on the synthesis of polymerizable amino acid derivatives and their incorporation into a polymer-based biohybrid interface to construct generic bioinspired analytical tools. We also utilized polyvinyl alcohol (PVA) as a sacrificial polymer to adjust the porosity of these biohybrid interfaces. The surface morphologies of the interfaces on gold electrodes were characterized by using scanning electron (SEM) and atomic force (AFM) microscopies. The electrochemical behavior of the polymeric films was systematically investigated using differential pulse voltammetry (DPV) to demonstrate the high affinity of the biohybrid interfaces for Cu(II) ions. The presence of macropores also significantly improved the recognition performance of the interfaces while enhancing interactions between the target [Cu(II) ions] and the functional groups. As a final step, we showed the applicability of the proposed analytical platform to create a Cu(II) ion-mediated supramolecular self-assembly on a quartz crystal microbalance (QCM) electrode surface in real time. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Bioinspiration and biomimicry are two recent materials-design concepts that have facilitated the creation of novel synthetic biomaterials for several applications, such as imaging, sensing, adsorption, catalysis and multi-therapy in biotechnology, as well as improving understanding of the underlying mechanisms of biological systems [1,2]. The recognition features of principal biocomponents such as amino acids, peptides and nucleic acids, which are responsible for the selective binding of many extended biomolecules in living systems, can be transferred to novel synthetic polymers by using a biomimetic approach. The insertion of functional parts (or active sites) or whole biomolecules into the backbone of polymeric structures is one of the possible ways to achieve this [3]. It is also well known that amino acid residues are fundamental for the functional properties of peptides, proteins and enzymes [4]. The amino acid sequence and arrangement through
∗ Corresponding author at: Hacettepe University, Department of Chemistry, Biochemistry Division, 06381-Beytepe, Ankara, Turkey. E-mail addresses: [email protected], [email protected] (L. Uzun). http://dx.doi.org/10.1016/j.snb.2017.05.030 0925-4005/© 2017 Elsevier B.V. All rights reserved.
polypeptide chains, regulates the binding sites of proteins for their ligands, the structural and surface properties of protein for folding as well as the catalytic activity of proteins (enzymes). Even small variations in amino acid sequence can significantly alter catalytic activity and ligand binding features, due to molecular interactions through non-covalent bonds, including hydrogen bonds, ionic interactions, van der Waals forces and hydrophobic interactions [5]. Amino acids play a major role in these interactions between ligands and biomolecules [6,7]. Amino and carboxylate moieties of amino acids serve as the chelating agent (N, O chelation) with various metal ions [7–11]. In addition, the imidazole ring of histidine, the phenol ring of tyrosine and the thiol group of cysteine, found in the binding sites of proteins, selectively form coordination complexes with metal ions [7]. This knowledge has led to a selective chromatographic method known as immobilized metalaffinity chromatography for protein and peptide purification under mild conditions [12–14]. Not only does the integration of recognition capability into macromolecules depend on the combination of these simple intermolecular interactions, but also the use of specific and selective counter molecules to construct supramolecular assemblies mainly relies on these non-covalent forces i.e. hydrogen bonding,
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hydrophobic, electrostatic, – and van der Waals interactions. In this context, supramolecular assembly of biological molecules, such as amino acids [15], peptides [16], proteins [17] and DNA [18] have been extensively explored for the design and development of novel functional biomaterials due to their specific recognition ability for target molecules. DNA-based supramolecular assemblies in particular have been studied and a construction strategy known as DNA origami is now well established [19]. The large number of possible combinations and the extremely wide variety of biomolecular building blocks have driven scientists from a diverse research spectrum including chemistry, biology, materials science, bioengineering and medicine, to focus their efforts on developing novel functional supramolecular assemblies for different biomaterial applications [20,21]. In this study, we have sought to integrate biological interactions into a generic supramolecular assembly as the basis for a smart and ready-to-use functional polymeric structure. Herein, we used N-methacryloyl-l-histidine (MAH) and N-methacryloyl-l-cysteine (MAC), which are the polymerizable derivatives of histidine and cysteine, respectively. MAC was proposed as self-assembling functional monomer for gold surfaces, which avoided the need to work with toxic thiol chemistry, while MAH functional monomer was proposed as a functional monomer with high affinity against Cu(II) ions because of its imidazole ring, to construct a metalion mediated supramolecular assembly. So, MAH was polymerized with 2-hydroxyethyl methacrylate (HEMA) in presence of polyvinyl alcohol (PVA) on a gold surface. PVA was introduced as a sacrificial polymer to obtain a highly porous polymeric structure in order to increase the intensity of response as well as to increase the accessibility of analyte molecules to electrode surfaces. After electrochemical characterization, the polymeric thin film was used to examine supramolecular self-assembling in real time using a mass-sensitive quartz crystal microbalance (QCM). 2. Experimental 2.1. Materials l-histidine, l-cysteine, poly(vinyl alcohol) (PVA), 2hydroxyethyl methacrylate (98%) (HEMA), ethylene glycol dimethacrylate (EGDMA), ␣,␣’-azoisobutyronitrile (AIBN), dimethyl sulfoxide (≥99.9%) (DMSO), sodium hydroxide (NaOH), ethyl acetate, 1,4-dioxane, copper(II) nitrate hemi(pentahydrate), IgG from human serum reagent grade (≥95% (HPLC) buffered aqueous solution and 1H-benzotriazole were supplied by Sigma Chemical Co. (St. Louis, USA). Aqueous solutions were prepared with Milli-Q deionized water (DI water, 18.2 cm) obtained using a Millipore system (Billerica, MA, USA). 2.2. Synthesis of amino acid-based functional monomers Amino acid based functional monomers, N-methacryloyll-histidine (MAH) and N-methacryloyl-l-cysteine (MAC) were prepared as previously described with minor modifications [22]. 5.52 mmol of amino acid (l-histidine or l-cysteine) was dissolved in 1 M aqueous solution of NaOH in a round-bottom flask. A solution of benzotriazole methacrylate (MA-Bt) (5.52 mmol) in 25 mL of 1,4-dioxane was slowly added to the amino acid solution and the reaction allowed to proceed at room temperature for 30 min with continuous stirring. After the reaction was complete, the 1,4dioxane was evaporated under vacuum. The residue was diluted with DI water and extracted with ethyl acetate 3 times (3 × 50 mL) to remove 1H-benzotriazole. The water solution was acidified to pH 6–7 and then the water was removed via rotary evaporation to obtain MAH.
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2.3. Design of sensor surfaces 2.3.1. Cleaning the gold coated substrate Fabrication of the polymeric films for electrochemical and FTIRATR characterization was performed on Au coated silica substrates (0.5 cm2 ). Substrates were produced in house by sputtering 2000 Å of Au onto a Ti coated (100 Å) adhesive layer on a silica wafer. Prior to use, the surface of the silica substrates was cleaned 5 times with alkaline piranha solution (H2 O:NH3 :H2 O2 (5/1/1, v/v)) at 80 ◦ C for 5 min. Following this, the samples were washed with excess DI water and pure ethyl alcohol and dried in a vacuum oven (200 mmHg, 40 ◦ C) for 2 h. After the cleaning process, 200 L of aqueous solution of MAC (5 mg/mL) were drop-cast onto the gold surfaces and left to react for 12 h in a sealed container, in order to form a self-assembled monolayer containing polymerizable functional groups of methacrylate that serve as grafting layer for the bulk polymer. Unbound MAC was removed by washing the surface with DI water and then, once more, the modified surfaces were dried in vacuum oven (200 mmHg, 25 ◦ C). 2.3.2. Synthesis of porous polymeric film with amino acid functionality Fabrication of the polymeric film having amino acid functionality and a macroporous structure was performed as follows: three stock solutions were separately prepared as monomer mixture, initiator, and pore-maker solutions. HEMA (1 mL), EGDMA (0.2 mL), DI water (0.25 mL) and MAH (5 mg) were mixed to prepare a stock monomer solution. The initiator stock solution included AIBN (2 mg/mL) in DMSO whereas pore-maker solution included PVA (25 mg/mL) in DI water. After that, a 10.0 L aliquot of the premixed monomer solution containing the first stock solution (100 L), PVA solution (20 L) and AIBN solution (40 L), was dropped onto the MAC-modified gold surface. Polymerization was initiated by raising the temperature to 70 ◦ C on a hot plate and continued for 5 min (Scheme 1). After the polymerization process, the sensor was washed with DI water at room temperature for 1 h, in order to remove sacrificial PVA and to expose the macropores created in the polymeric structure. To assess the effects of macroporosity and the amino acid moieties in the polymeric structure, three different combinations were examined by varying the presence/absence of amino acid-based monomer (MAH) and, for control purposes, the sacrificial polymer (PVA): (i) amino acid functionality + macroporosity; (ii) amino acid functionality; (iii) macroporosity. The first combination had all functionalities, the second one had only histidine groups in a non-porous polymeric block, and the third one only had a basic macroporous structure. 2.3.3. Synthesis of porous polymeric film on QCM electrodes QCM measurements were used to monitor the Cu(II)-mediated supramolecular assembling of biomolecules in real time. QCM Au electrodes were coated with polymeric film having amino acid functionality and porous structure, following the protocol described in Section 2.3.2 (Scheme 1). 2.4. Selectivity and reversibility In order to show the selectivity and reversibility of the formation of supramolecular assembly, three different sets of experiments were performed by applying impedance measurements as mentioned before. Herein, bias potential, frequency range, amplitude, redox probe in the solution and its concentration were adjusted as 0.2 V, 0.1 Hz–0.1 MHz, 5 mV, and 10 mM [Fe(CN)6 ]3−/4− in 0.1 M KCl(aq), respectively. In the first set, metal ion coordination was performed with three different ions [Cu(II), Zn(II), and Pb(II)] at four different concentrations. In the second set, the orientation of four different biomolecules (albumin, immunoglobulin, transferrin, and
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Scheme 1. Schematic showing: (a) synthesis of functional monomer, and (b) creation of porous polymeric network. –1- Self-assembling of cysteine-based monomer and introduction of monomer solution, –2- thermal polymerization to create a porous structure with imidazole (histidine) functionalities, –3- Cu(II) chelation onto imidazole ring, and –4- human immunoglobulin orientation to form supramolecular assembly. The imidazole ring is mainly responsible for chelating Cu(II) ions while water molecules complete the coordination vacancies of Cu(II) ions, which are replaced with protein molecules during the supramolecular assembly formation.
anti-transferrin antibody) on the polymeric films was evaluated before and after Cu(II) ions coordination. Herein, the formation of supra-molecular assemblies between immunoglobulin/transferrin and anti-transferrin/transferrin pairs is also evaluated. In the third set, the elution/destruction of the supramolecular assemblies was investigated by using sodium chloride (NaCl) and ethylenediamine tetra acetic acid (EDTA) as eluting agents. NaCl was chosen to destroy the interaction between Cu(II) ions and the biomolecules and Na4 EDTA was chosen to destroy the interaction between Cu(II) ions and the polymeric films directly.
2.5. Instrumentation All voltammetry measurements were carried out using an Ivium Stat.XR electrochemical analyzer (Ivium Tech., Eindoven, Netherlands). A three-electrode cell with gold coated silica substrate as a working electrode, platinum wire as an auxiliary and an Ag/AgCl (3M KCl) reference electrode were used for the voltammetric measurements. A Q-Sense QCM-D Technology (Biolin Scientific Holding AB, Stockholm, Sweden) device was used for the QCM measurements. 5 MHz, 14 mm diameter, polished, ATcut, gold electrodes were used in these experiments. Atomic force microscopy (AFM) observations were carried out using ambient AFM (Nanomagnetics Instruments, Oxford, UK) in tapping mode in an air atmosphere. The AFM experimental parameters were: oscillation frequency (341.30 Hz), vibration amplitude (1VRMS ) and free vibration amplitude (2VRMS ). Samples were scanned at a 1 m/s scanning rate and 256 × 256 pixels resolution to obtain a view of a 2 m × 2 m area. Scanning electron microscopy (SEM) observations were performed using a LEO 155 Gemini (Zeiss, OR, USA). FTIR-ATR measurements were carried out using a Bruker Vertex 70 FTIR spectrometer (PerkinElmer, Waltham, MA, USA).
3. Results and discussion 3.1. Preparation and characterization of polymeric film on gold surfaces The morphology of the polymeric films (supported on the gold surface) was characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The presence of the functional groups in the polymeric films was confirmed using a FTIR spectrometer. The electrochemical behavior of the polymeric films was systematically investigated using differential pulse voltammetry (DPV). Fig. 1a–c shows the SEM images of polymeric films produced in the presence (Fig. 1a,b) and the absence (Fig. 1c) of PVA. As can be seen from Fig. 1a–b, the presence of PVA as a sacrificial polymer resulted in structures with different porosity, as expected. Washing the polymeric film with DI after polymerization exposed large pores of various sizes, which were randomly distributed in the body of the polymer. It has previously been reported that the distribution and size of the pores can be controlled by varying the concentration of sacrificial polymers (i.e. PVA, polyvinylpyridine) to adjust the pore size during copolymerization [23,24] but this was not explored here, since we focused on enhanced amino acid functionality rather than tuning the pore size. The absence of PVA caused relatively non-porous structures (Fig. 1c) (extra SEM images at different magnification were given in Supporting Information file, Figs. S1–S3). These findings were confirmed by the AFM images (Fig. 2a,b). As can be seen in the figure, the presence of PVA directly affected the surface morphology and porosity, while the resulting pores were from nano- to microscale. In order to confirm the presence and efficient removal of PVA from the polymeric network, as well as the chemical structure of the polymer, FTIR-ATR
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Fig. 1. SEM images of polymeric films: (a) with porous structure having amino acid functionality, (b) porous structure without amino acid functionality, and (c) only amino acid functionality.
Fig. 2. AFM images of polymeric films with porous structure having: (a) amino acid functionality, and (b) non-porous structure with amino acid functionality.
measurements were performed (Fig. 3). The bands around frequencies 1617 (C O, amide I) and 1470 cm−1 (N H bending, amide II) stemmed from the functional monomer, MAH and indicated its successful incorporation into the polymeric structure. After washing the polymer with DI, the dominant IR bands at 1554 and 1410 cm−1 arising from the carboxylate (87–90% hydrolyzed) and methylene ( CH2 ) groups of PVA disappeared. Also, the intensity of the band at 3310 cm−1 for alcohol ( OH) groups decreased after washing.
These results were consistent with the presence of PVA in the polymeric structure as a sacrificial polymer via physical adsorption and its successful removal after an efficient washing step [25]. The synthesis of a monomer with amino acid functionality and its incorporation into the polymeric structure is one, and probably the easiest, of the possible ways to integrate the biorecognition mechanism of biomolecules into biomimicking artificial networks. It is also well known that amino acid residues are the biologi-
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Fig. 3. FTIR-ATR spectra of the functional polymer (with porous structure having amino acid functionality) before and after removal of PVA.
cal building blocks for the selective substrate binding in a diverse range of biomolecules, such as proteins, enzymes etc. Therefore, we aimed to synthesize a polymerizable amino acid (l-histidine) derivative (named MAH) through a benzotriazole-mediated reaction (Scheme 1).
Differential pulse voltammetry (DPV) was used to evaluate the presence of histidine molecules in the polymeric film and their ability to coordinate Cu(II) ions due to the Lewis base character of the nitrogen atoms in the hetero-aromatic ring (imidazole moieties). The first step of the investigation was to study the effects of pH and incubation time on the chelation between the Cu(II) ions and the imidazole ring [26,27]. The pH values were varied by using different buffer systems in the pH range 4.0–7.4. We determined the optimal pH for Cu(II) ion chelation as 5.0 (Fig. 4a), where the nitrogen atoms have unpaired electrons in the imidazole ring and therefore are much more accessible for efficient Cu(II) ion chelation. At more alkaline pH values (pH > 7.0), a significant reduction in the electrochemical response was recorded; this could be due to the formation of the metal-oxide (CuO, Cu2 O etc) [28] and a subsequent reduction in capture efficiency. The chelation reaction rapidly reached a plateau value after about 120 s (Fig. 4b). After 120 s, the change in current peak was no longer significant and so 120 s was used as incubation time for further measurements. The results indicated that the adsorption kinetics of the polymeric film having both amino acid functionality and porous structure were quite fast and efficient. In order to understand the effect of the amino acid functionalities and porosity on the Cu(II) ion chelation, we synthesized three different polymers and compared their performance: (i) with both amino acid functionality and pores (PF1); (ii) with only amino acid functionality (PF2); and (iii) with only pores/without amino acid functionalities (PF3). Calibration curves were recorded with the different polymeric films over a Cu(II) ion concentration range of 0–1000 nM (Fig. 4d). As can be seen in Fig. 4c, the increase in Cu(II) ion concentration resulted in
Fig. 4. Differential pulse voltammetry responses of polymeric film having amino acid functionality and porous structure with: (a) various pH at 10 mV/s scan rate vs Ag/AgCl reference electrode, (b) effect of incubation time in acetate buffer pH: 5.0 (10 mM) at 10 mV/s scan rate vs Ag/AgCl refer-ence electrode, (c) analytical response of the modified electrode with increasing concentration of Cu, and (d) calibration curve.
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Fig. 5. Differential pulse voltammetry responses of three electrodes (PF1, with both amino acid functionality and pores; PF2, with only amino acid functionality; and PF3, with only pores/without amino acid functionalities) in acetate buffer (pH: 5.0, 10 mM) at 10 mV/s scan rate vs Ag/AgCl reference electrode.
an increase in the current peak for PF1. Due to the presence of amino acid functionality, PF2 also responded to the increasing concentration of Cu(II) ions, but the responsiveness of PF2 was much lower than that of PF1(see also Supporting Information Fig. S4). This result indicated that not only amino acid functionality, but also the film porosity has a synergic effect on controlling the recognition ability of the polymeric films. On the other hand, the polymeric film having only porous structure, showed small responses with respect to the presence of increasing concentration of Cu(II) (Fig. 5). This slight response could be due to the physical/chemical adsorption of the metal ions into the polymeric network (see also Supporting Information Fig. S5). As mentioned before, these results emphasized successful incorporation of the functional monomer, MAH into the backbone poly(HEMA), while imparting a macroporous structure to the polymeric film. 3.2. Supramolecular self-assembly on a QCM electrode surface
Fig. 6. Cu(II) ions captured on the gold surface of a QCM electrode coated with polymeric film having amino acid functionality and porous structure.
The main goal of this study was the integration of the biorecognition ability of the amino acid onto an easy-to-use sensing platform. On the basis of the results obtained for characterization and electrochemical evaluation reported above, we applied the same procedure to create functional polymeric films on the gold-coated surface of QCM crystals and to utilize their ability to form selfassembled supramolecular structures. Two different approaches were adopted: (i) time course real-time Cu(II) immobilization; and (ii) Cu(II)-mediated immunoglobulin G (IgG) orientation on the QCM chip. For the first, IgG was chosen as a model protein to optimize the parameters and compare the efficiencies of PF1 and PF2 films. In the second approach, we aimed to show how this approach might be utilized for forming a supramolecular assembly as a biosensor platform. The effect of Cu(II) ion concentration over the range 0.1–10 mg/mL, in acetate buffer (pH: 5.0, 10 mM) at constant flow rate 10 L/min, was evaluated (Fig. 6). The frequency shifts of the QCM were monitored and calculated using QTools software (Stockholm, Sweden). The QCM chip was exposed to Cu(II) ion solution at four different concentrations, followed by desorption and regeneration achieved using ethylenediamine tetraacetic acid (EDTA, 25 mM) solution and acetate buffer (pH 5.0), respectively. Fig. 7 illustrates that all steps, including equilibration–adsorptiondesorption-regeneration, were rapidly and reversibly completed in about 30 min. PF1 film on the QCM chip showed a linear
response over this concentration range with a very high regression coefficient (R2 ) of 0.9911. These results indicated the successful integration of this approach with the QCM while retaining the recognition abilities. The next step was to demonstrate the efficiency of Cu(II) mediated IgG recognition/orientation, which is a cornerstone of our main goal to develop supramolecular assemblies for biosensing platforms. For this purpose, we interacted IgG solutions over a concentration range of 0.5 and 100 g/mL (phosphate buffer, pH: 7.4, 10 mM) with a PF1-modified QCM chip with and without the Cu(II) ion chelation step, at a constant flowrate of 10 L/min (Fig. 7). Fig. 7a shows that the PF1 QCM chip responded to the increase in IgG concentration with a regression coefficient (R2 ) of 0.8657 (with a linear response of 86.6%) due to the specific interaction between histidine and IgG molecules [29,30]. However, the frequency shift and linearity improved with respect to regression coefficient (R2 : 0.9056 with a linear response of 90.56%) after Cu(II) chelation and delta frequency values decreased from 5.21 to 84.16 at an IgG concentration of 100 g/mL (Fig. 7b). The Cu(II) ion-mediated IgG orientation significantly affected the amount of IgG molecules immobilized (Fig. 7c), because of the specific interaction between Cu(II) ions and IgG which occurs through the Fc-fragment [31]. We should also mention here that all steps, including the equilibration-adsorption (six different concen-
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phate buffer (pH: 7.4, 10 mM), which indicated good reusability and cost-efficiency of the supramolecular assemblies. 3.3. Selectivity and reversibility As mentioned above, three different sets of impedance measurement experiments were performed (see Supporting Information Fig. S6 for EES circuit used). In order to allow for the variations in response of polymeric films due to film structure, metal ions and biomolecules, we converted the data into the normalized responses which were calculated as follows: R/R o = (R f − R o )/R o
Fig. 7. Supramolecular self-assembling on the gold surface QCM electrode coated with polymeric film having amino acid functionality and porous structure: (a) IgG assembling in absence of Cu(II) ions, (b) IgG assembling mediated via Cu(II) ions, (c) comparison of two systems.
trations) and desorption–regeneration were completed in about 100 min, which meant that each concentration reached equilibrium in about only 12 min. Also, we observed successful regeneration of the QCM chip under mild conditions by using 1 M NaCl in phos-
(A.1)
where Rf and Ro were the responses of electrodes after and before treatment with each analyte, respectively. As seen in Fig. 8a, Cu(II) ion coordination enhanced the biomolecule orientation on the polymeric films, especially in the cases of IgG and transferrin. This indicated an increase in affinity of biomolecules to adsorb onto surface (see also Supporting Information Figs. S7 and S8). The electrodes investigated, PF2 resulted in the highest responses for all biomolecules before and after Cu(II) ion coordination, due to the non-porous structure providing greater insulation, and hence higher resistivity. The transferrin orientation on Cu(II) ions showed the highest increase in responses because of its metal binding pocket. The increases were 8.17-, 5.91-, and 1.60- folds for PF1, PF2, and PF3 electrodes, respectively. These results also confirmed both successful Cu(II) ion coordination and the porous structure of the polymeric films as well as the incorporation of metal chelating groups (histidine in MAH monomer) into the polymeric backbone. Note that only PF1 included the porous structure and MAH groups. In addition to biomolecule orientation on Cu(II) ions, the formation of supramolecular assemblies between the recognizing biomolecules (IgG molecule and anti-transferrin antibody) and the target molecule (transferrin) was also monitored both in the presence and absence of Cu(II) ions (Fig. 8b). In this respect, we aimed to investigate how Cu(II) plays a major role during the supramolecular assembly formation process (see also Supporting Information Figs. S9 and S10). In the absence of Cu(II) ions, the responses were inhibited by addition of a second biomolecule (transferrin), which indicated competition and replacement of transferrin with the other biomolecules. Therefore, the responses were lower than those for both of recognizing biomolecules and transferrin. In the case of Cu(II) ion coordination, the interaction between the recognizing biomolecule and Cu(II) ions was protected and so the formation of supramolecular assembly was possible. The increases in the responses for IgG/transferrin pair were, in presence of Cu(II) ions, calculated as 3.41-, 3.92-, and 1.70-fold, whereas that for the anti-transferrin/transferrin pair were calculated as 7.50-, 3.41-, and 5.11-fold for PF1, PF2, and PF3 electrodes, respectively. The highest increase exhibited by PF1 indicated the high affinity of the electrodes resulting from the porous structure and histidine functionality as well as confirming the formation of a supramolecular assembly (see also Supporting Information Fig. S11 and S12). Fig. 8c showed the effects of the type and concentration of metal ions, which was another set of experiments to confirm ion coordination depending on the structural (porous vs. non-porous) and chemical (having histidine or not) features of the polymeric film. As seen in the figure, PF1 showed the most dependency on the type of metal ions and the least dependency on their concentration. These results indicated that: (i) metal ions were coordinated by histidine groups, (ii) metal ion coordination decreased permeability of the probe couple by blocking the pores in the polymeric structure, and (iii) the increase in the metal ion concentration did not effect on the response due to occupation of histidine functionalities. The non-porous structure of PF2 decreased the sensitivity of
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Fig. 8. The selectivity and reversibility of the formation of supramolecular assembly. (a) the effect of Cu(II) ion coordination on the selectivity of orientation of four different biomolecules, (b) the effects of Cu(II) coordination and the recognizing biomolecule on the formation of supramolecular assembly, (c) the effects of the type and concentration of metal ions, and (d) the reversibility of interactions between Cu(II) ions/biomolecules and Cu(II) ions/polymeric film. Herein, bias potential, frequency range, amplitude, redox probe in the solution and its concentration were adjusted as 0.2 V, 0.1 Hz–0.1 MHz, 5 mV, and 10 mM [Fe(CN)6 ]3−/4− in 0.1 M KCl(aq), respectively.
the polymeric film to the type of metal ions. However, the increase in the concentration caused significant variation in the responses due to the coordination of metal ions by histidine functionalities on the surface. In the case of PF3, both of the type and concentration of metal ions caused a considerable variation in the responses. In the third set of experiments, we aimed to show the reversibility of the formation of the supramolecular assembly; so the selective elution of only biomolecules (protecting Cu(II) coordination) and Cu(II) ions (the complete destruction of assembly) was evaluated by using NaCl and EDTA as the eluting agents (Fig. 8d). As seen in the figure, NaCl treatment helped to remove biomolecular assembly between anti-transferrin and transferrin while protecting the Cu(II) coordination on the PF1 film (see also impedance response of Cu(II) ion coordination in Supporting Information, Fig. S13). However, EDTA treatment resulted in a response the same as the plain PF1 electrode, which indicated a direct destruction of the supramolecular assembly through chelating of metal ions by means of EDTA molecules. We should mention that each step (NaCl and EDTA treatments) was performed separately after the formation of the supra molecular assembly. The results confirmed the selectivity and reversibility of the formation of supramolecular assemblies while illustrating the considerable potential of the materials developed for the easy design of new analytical biosensors.
4. Conclusions Biological elements such as amino acids, nucleotides, and monosaccharides, play a major role in biorecognition of biological molecules. Sometimes, a single change in sequence results in completely different biorecognition abilities. Histidine especially, as one of the more active functional amino acids, has a role in metal ion coordination and intermolecular communications. Inspired by this knowledge, we focused our attention on developing a biosensing platform through simple, efficient and selective supramolecular assembly. We synthesized polymerizable histidine derivatives to directly insert them into a porous polymeric network and to integrate their biorecognition capabilities into artificial surfaces. Histidine incorporation into the polymeric structure allowed faster recognition of Cu(II) ions (only 120 s) over a wide concentration range of 0–1000 nM. In addition, this enhanced the Cu(II)-ion mediated IgG recognition/orientation process and resulted in significant improvement in the linear regression coefficients obtained. We also demonstrated how this strategy could be used to construct supramolecular assemblies as recognition elements in biosensing platforms. The successful QCM results revealed that this simple approach should be applicable to a variety of sensing platforms. Also, the basic idea of metal ion-mediated supramolecular assembly formation, is very useful for developing an efficient biohybrid sensing interface with an improved response time and signal inten-
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sity as well as better linearity, which increased from a correlation coefficient of 0.8657–0.9056 as a consequence of Cu(II) ion chelation with histidine groups. In conclusion, the results demonstrate that this approach is simple, easy-to-use, selective, reversible, and reusable and should be applicable to different sensing platforms as an inexpensive way to develop efficient biohybrid sensing platforms. Acknowledgements The authors wish to acknowledge the Swedish Research Council (VR-2011-6058357) for generous financial support for this research. L. Uzun thanks to the European Commission for Marie Curie fellowship (MIFs4BioMed) with grant agreement number: 629251. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.05.030. References [1] C. Sanchez, H. Arribart, M. Madeleine, G. Guille, Biomimetism and bioinspiration as tools for the design of innovative materials and systems, Nat. Mater. 4 (2005) 277–288. [2] G.M. Whitesides, Bioinspiration: something for everyone, Interface Focus 5 (2015) 1–10. [3] M. Hussain, J. Wackerlig, P.A. Lieberzeit, Biomimetic strategies for sensing biological species, Biosensors 3 (2013) 89–107. [4] R. Vaidhyanathan, D. Bradshaw, J. Rebilly, J.P. Barrio, J.A. Gould, N.G. Berry, M.J. Rosseinsky, A family of nanoporous materials based on an amino acid backbone, Angew. Chem. Int. Ed. 45 (2006) 6495–6499. [5] B. Alberts, A. Johnson, J. Lewis, M. Raft, K. Roberts, P. Walter, Molecular Biology of the Cell, fourth ed., Garland Science, New York, USA, 2002. [6] A.N. Jha, S. Vishveshwara, J.R. Banavar, Amino acid interaction preferences in proteins, Protein Sci. 19 (2010) 603–616. [7] Y. Shimazaki, M. Takani, O. Yamauchi, Metal complexes of amino acids and amino acid side chain groups. Structures and properties, Dalton Trans. 38 (2009) 7854–7869. [8] S.H. Laurie, Comprehensive Coordination Chemistry, Pergamon Press, Oxford, 1987. [9] S.H. Laurie, Handbook of Metal–Ligand Interactions in Biological Fluids: Bioinorganic Chemistry, Marcel Dekker, New York, USA, 1995. [10] T. Kiss, Biocoordination Chemistry: Coordination Equilibria in Biologically Active Systems, Ellis Horwood, Chichester, UK, 1990. [11] E. Chow, J.J. Gooding, Peptide modified electrodes as electrochemical metal ion sensors, Electroanal 18 (2006) 1437–1448. [12] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Metal chelate affinity chromatography, a new approach to protein fractionation, Nature 258 (1975) 598–599. [13] E.B. Altintas, N. Tuzmen, L. Uzun, A. Denizli, Immobilized metal affinity adsorption for antibody depletion from human serum with monosize beads, Ind. Eng. Chem. Res. 46 (2007) 7802–7810. [14] A.A. Ozcan, R. Say, A. Denizli, A. Ersoz, L-Histidine imprinted synthetic receptor for biochromatography applications, Anal. Chem. 78 (2006) 7253–7258. [15] E. Rosini, G. Molla, C. Rossetti, M.S. Pilone, L. Pollegioni, S. Sacchi, A biosensor for all d-amino acids using evolved d-amino acid oxidase, J. Biotechnol. 135 (2008) 377–384. [16] G.J. Zhang, M.J. Huang, J.J. Ang, E.T. Liu, K.V. Desai, Self-assembled monolayer-assisted silicon nanowire biosensor for detection of protein–DNA interactions in nuclear extracts from breast cancer cell, Biosens. Bioelectron. 26 (2011) 3233. [17] J. Luo, Q.D. Zhang, Y.Y. Huang, G.Q. Liu, R. Zhao, Quartz crystal microbalance biosensor for recombinant human interferon- detection based on antisense peptide approach, Anal. Chim. Acta. 590 (2007) 91–97. [18] J. Zhang, H.L. Qi, Y. Li, J. Yang, Q. Gao, C.X. Zhang, Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex, Anal. Chem. 80 (2008) 2888–2894. [19] Q. Jiang, Y. Shi, Q. Zhang, N. Li, P. Zhan, L. Song, L. Dai, J. Tian, Y. Du, Z. Cheng, B. Ding, A self-assembled DNA origami-gold nanorod complex for cancer theranostics, Small 38 (2015) 5134–5141.
[20] N. Stephanopoulos, J.H. Ortony, I.S. Stupp, Self-assembly for the synthesis of functional biomaterials, Acta Mater. 61 (2013) 912–930. [21] L.P. Xu, Y. Liu, X. Zhang, Interfacial self-assembly of amino acids and peptides: scanning tunneling microscopy investigation, Nanoscale 3 (2011) 4901–4915. [22] D. Hür, S.F. Ekti, R. Say, N-acylbenzotriazole mediated synthesis of some methacrylamido amino acids, Lett. Org. Chem. 4 (2007) 585–587. [23] W. Lee, X. Zhang, R.M. Briber, A simple method for creating nanoporous block- copolymer thin films, Polymer 51 (2010) 2376–2382. [24] I. Tokarev, S. Minko, Stimuli-responsive porous hydrogels at interfaces for molecular filtration separation, controlled release, and gating in capsules and membranes, Adv. Mater. 22 (2010) 3446–3462. [25] P.J. Larkin, Infrared and Raman Spectroscopy Principles and Spectral Interpretation, Elsevier, Amsterdam, Netherland, 2011. [26] W. Yang, E. Chow, G.D. Willett, D.B. Hibbert, J.J. Gooding, Exploring the use of the tripeptide Gly–Gly–His as a selective recognition element for the fabrication of electrochemical copper sensors, Analyst 128 (2003) 712–718. [27] W. Yang, J.J. Gooding, D.B. Hibbert, Redox voltammetry of sub-parts per billion levels of Cu2+ at polyaspartate-modified gold electrodes, Analyst 126 (2001) 1573–1577. [28] M.M. Antonijevic, S.C. Alagic, M.B. Petrovic, M.B. Radovanovic, A.T. Stamenkovic, The influence of pH on electrochemical behavior of copper in presence of chloride ions, Int. J. Electrochem. Sci. 4 (2009) 516–524. [29] Application Note 127: Separation of an Intact Monoclonal Antibody and Fractionation of Monoclonal Antibody Papain Digest Fragments Using Immobilized Metal Affinity Chromatography (IMAC), Dionex Corporation, Sunnyvale, CA, 2006. [30] S. Asliyuce, L. Uzun, R. Say, A. Denizli, Immunoglobulin G recognition with Fab fragments imprinted monolithic cryogels: evaluation of the effects of metal-ion assisted-coordination of template molecule, React. Funct. Polym. 73 (2013) 813–820. [31] G. Erturk, L. Uzun, M.A. Tumer, R. Say, A. Denizli, Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection, Biosensor. Bioelectron. 28 (2011) 97–104.
Biographies Erdogan Ozgur received BS degree from department of chemistry education (2008) and MSc and PhD degrees in chemistry from Hacettepe University, Turkey (2011 and 2016). His research interests are molecular imprinted polymeric micro/nano materials, and their applications. Onur Parlak received his PhD degree in Bioelectronics from Linköping University, Biosensors and Bioelectronics Centre in September 2015. Earlier, he was visiting research intern at Nanyang Technological University, Materials Science and Engineering Department, Singapore in 2011. He received his master and bachelor degree from Izmir Institute of Technology, Department of Chemistry in 2011 and 2009, respectively. He was recently awarded by one of the most prestigious grant in Sweden, Knut Allice Wallenberg Foundation for postdoctoral research fellow at Stanford University, Materials Science and Engineering, working with Prof. Alberto Salleo, currently. Valerio Beni works with development of electrochemical chemo/bio-sensors, for environmental, clinical and food analysis, and their integration with hybrid-printed platform. Previously, Beni has worked as a Postdoctoral Researcher at the Irish Tyndall National Institute, a Marie Curie Fellow at the Universitat Rovira I Virgili in Spain and as an Assistant Professor at Linköping University. Beni holds a Ph.D. in Chemistry. His core expertise lies in the area of electrochemical chemo/bio-sensors and their applications in clinical, food and environmental analysis. Anthony P.F. Turner is currently Head of Division FM-Linköping University’s new Centre for Biosensors and Bioelectronics. His previous thirty-five year academic career in the UK culminated in the positions of Principal (Rector) of Cranfield University and Distinguished Professor of Biotechnology. Professor Turner has more than 600 publications and patents in the field of biosensors and biomimetic sensors and is best known for his role in the development of glucose sensors for home-use by people with diabetes. He published the first textbook on biosensors in 1987 and is Editor-In-Chief of the principal journal in his field, Biosensors & Bioelectronics, which he co-founded in 1985. Lokman Uzun is an Associate Professor at the Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey where he also received his PhD in 2008. He is the author of more than 100 articles in peer-review journals with a h-index value of 27 and one of Associate Editors of Advanced Materials Letters. He recently awarded by an European Union Marie-Curie fellowship with the Biosensors and Bioelectronics Centre, Linköping University, Sweden for two years. His research interest is mainly in materials science, surface modification, affinity interaction, polymer science, especially molecularly imprinted polymers and their applications in biosensors, bioseparation, food safety, and the environmental sciences.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Bioinspired design of a polymer-based biohybrid sensor interface Erdo˘gan Özgür a,b , Onur Parlak b , Valerio Beni b,c , Anthony P.F. Turner b , Lokman Uzun a,b,∗ a
Hacettepe University, Department of Chemistry, Ankara, Turkey Linköping University, Biosensors and Bioelectronics Centre, IFM, Linköping, Sweden c RISE Acreo, Research Institute of Sweden, Norrköping, Sweden b
a r t i c l e
i n f o
Article history: Received 24 January 2017 Received in revised form 23 April 2017 Accepted 8 May 2017 Available online 10 May 2017 Keywords: Biomimicry Amino acid Macroporosity Polymeric film Supramolecular self-assembly
a b s t r a c t The key step in the construction of efficient and selective analytical separations or sensors is the design of the recognition interface. Biomimicry of the recognition features typically found in biological molecules, using amino acids, peptides and nucleic acids, provides plausible opportunities to integrate biological molecules or their active sites into a synthetic polymeric backbone. Given the basic role of functional amino acids in biorecognition, we focused on the synthesis of polymerizable amino acid derivatives and their incorporation into a polymer-based biohybrid interface to construct generic bioinspired analytical tools. We also utilized polyvinyl alcohol (PVA) as a sacrificial polymer to adjust the porosity of these biohybrid interfaces. The surface morphologies of the interfaces on gold electrodes were characterized by using scanning electron (SEM) and atomic force (AFM) microscopies. The electrochemical behavior of the polymeric films was systematically investigated using differential pulse voltammetry (DPV) to demonstrate the high affinity of the biohybrid interfaces for Cu(II) ions. The presence of macropores also significantly improved the recognition performance of the interfaces while enhancing interactions between the target [Cu(II) ions] and the functional groups. As a final step, we showed the applicability of the proposed analytical platform to create a Cu(II) ion-mediated supramolecular self-assembly on a quartz crystal microbalance (QCM) electrode surface in real time. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Bioinspiration and biomimicry are two recent materials-design concepts that have facilitated the creation of novel synthetic biomaterials for several applications, such as imaging, sensing, adsorption, catalysis and multi-therapy in biotechnology, as well as improving understanding of the underlying mechanisms of biological systems [1,2]. The recognition features of principal biocomponents such as amino acids, peptides and nucleic acids, which are responsible for the selective binding of many extended biomolecules in living systems, can be transferred to novel synthetic polymers by using a biomimetic approach. The insertion of functional parts (or active sites) or whole biomolecules into the backbone of polymeric structures is one of the possible ways to achieve this [3]. It is also well known that amino acid residues are fundamental for the functional properties of peptides, proteins and enzymes [4]. The amino acid sequence and arrangement through
∗ Corresponding author at: Hacettepe University, Department of Chemistry, Biochemistry Division, 06381-Beytepe, Ankara, Turkey. E-mail addresses: [email protected], [email protected] (L. Uzun). http://dx.doi.org/10.1016/j.snb.2017.05.030 0925-4005/© 2017 Elsevier B.V. All rights reserved.
polypeptide chains, regulates the binding sites of proteins for their ligands, the structural and surface properties of protein for folding as well as the catalytic activity of proteins (enzymes). Even small variations in amino acid sequence can significantly alter catalytic activity and ligand binding features, due to molecular interactions through non-covalent bonds, including hydrogen bonds, ionic interactions, van der Waals forces and hydrophobic interactions [5]. Amino acids play a major role in these interactions between ligands and biomolecules [6,7]. Amino and carboxylate moieties of amino acids serve as the chelating agent (N, O chelation) with various metal ions [7–11]. In addition, the imidazole ring of histidine, the phenol ring of tyrosine and the thiol group of cysteine, found in the binding sites of proteins, selectively form coordination complexes with metal ions [7]. This knowledge has led to a selective chromatographic method known as immobilized metalaffinity chromatography for protein and peptide purification under mild conditions [12–14]. Not only does the integration of recognition capability into macromolecules depend on the combination of these simple intermolecular interactions, but also the use of specific and selective counter molecules to construct supramolecular assemblies mainly relies on these non-covalent forces i.e. hydrogen bonding,
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hydrophobic, electrostatic, – and van der Waals interactions. In this context, supramolecular assembly of biological molecules, such as amino acids [15], peptides [16], proteins [17] and DNA [18] have been extensively explored for the design and development of novel functional biomaterials due to their specific recognition ability for target molecules. DNA-based supramolecular assemblies in particular have been studied and a construction strategy known as DNA origami is now well established [19]. The large number of possible combinations and the extremely wide variety of biomolecular building blocks have driven scientists from a diverse research spectrum including chemistry, biology, materials science, bioengineering and medicine, to focus their efforts on developing novel functional supramolecular assemblies for different biomaterial applications [20,21]. In this study, we have sought to integrate biological interactions into a generic supramolecular assembly as the basis for a smart and ready-to-use functional polymeric structure. Herein, we used N-methacryloyl-l-histidine (MAH) and N-methacryloyl-l-cysteine (MAC), which are the polymerizable derivatives of histidine and cysteine, respectively. MAC was proposed as self-assembling functional monomer for gold surfaces, which avoided the need to work with toxic thiol chemistry, while MAH functional monomer was proposed as a functional monomer with high affinity against Cu(II) ions because of its imidazole ring, to construct a metalion mediated supramolecular assembly. So, MAH was polymerized with 2-hydroxyethyl methacrylate (HEMA) in presence of polyvinyl alcohol (PVA) on a gold surface. PVA was introduced as a sacrificial polymer to obtain a highly porous polymeric structure in order to increase the intensity of response as well as to increase the accessibility of analyte molecules to electrode surfaces. After electrochemical characterization, the polymeric thin film was used to examine supramolecular self-assembling in real time using a mass-sensitive quartz crystal microbalance (QCM). 2. Experimental 2.1. Materials l-histidine, l-cysteine, poly(vinyl alcohol) (PVA), 2hydroxyethyl methacrylate (98%) (HEMA), ethylene glycol dimethacrylate (EGDMA), ␣,␣’-azoisobutyronitrile (AIBN), dimethyl sulfoxide (≥99.9%) (DMSO), sodium hydroxide (NaOH), ethyl acetate, 1,4-dioxane, copper(II) nitrate hemi(pentahydrate), IgG from human serum reagent grade (≥95% (HPLC) buffered aqueous solution and 1H-benzotriazole were supplied by Sigma Chemical Co. (St. Louis, USA). Aqueous solutions were prepared with Milli-Q deionized water (DI water, 18.2 cm) obtained using a Millipore system (Billerica, MA, USA). 2.2. Synthesis of amino acid-based functional monomers Amino acid based functional monomers, N-methacryloyll-histidine (MAH) and N-methacryloyl-l-cysteine (MAC) were prepared as previously described with minor modifications [22]. 5.52 mmol of amino acid (l-histidine or l-cysteine) was dissolved in 1 M aqueous solution of NaOH in a round-bottom flask. A solution of benzotriazole methacrylate (MA-Bt) (5.52 mmol) in 25 mL of 1,4-dioxane was slowly added to the amino acid solution and the reaction allowed to proceed at room temperature for 30 min with continuous stirring. After the reaction was complete, the 1,4dioxane was evaporated under vacuum. The residue was diluted with DI water and extracted with ethyl acetate 3 times (3 × 50 mL) to remove 1H-benzotriazole. The water solution was acidified to pH 6–7 and then the water was removed via rotary evaporation to obtain MAH.
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2.3. Design of sensor surfaces 2.3.1. Cleaning the gold coated substrate Fabrication of the polymeric films for electrochemical and FTIRATR characterization was performed on Au coated silica substrates (0.5 cm2 ). Substrates were produced in house by sputtering 2000 Å of Au onto a Ti coated (100 Å) adhesive layer on a silica wafer. Prior to use, the surface of the silica substrates was cleaned 5 times with alkaline piranha solution (H2 O:NH3 :H2 O2 (5/1/1, v/v)) at 80 ◦ C for 5 min. Following this, the samples were washed with excess DI water and pure ethyl alcohol and dried in a vacuum oven (200 mmHg, 40 ◦ C) for 2 h. After the cleaning process, 200 L of aqueous solution of MAC (5 mg/mL) were drop-cast onto the gold surfaces and left to react for 12 h in a sealed container, in order to form a self-assembled monolayer containing polymerizable functional groups of methacrylate that serve as grafting layer for the bulk polymer. Unbound MAC was removed by washing the surface with DI water and then, once more, the modified surfaces were dried in vacuum oven (200 mmHg, 25 ◦ C). 2.3.2. Synthesis of porous polymeric film with amino acid functionality Fabrication of the polymeric film having amino acid functionality and a macroporous structure was performed as follows: three stock solutions were separately prepared as monomer mixture, initiator, and pore-maker solutions. HEMA (1 mL), EGDMA (0.2 mL), DI water (0.25 mL) and MAH (5 mg) were mixed to prepare a stock monomer solution. The initiator stock solution included AIBN (2 mg/mL) in DMSO whereas pore-maker solution included PVA (25 mg/mL) in DI water. After that, a 10.0 L aliquot of the premixed monomer solution containing the first stock solution (100 L), PVA solution (20 L) and AIBN solution (40 L), was dropped onto the MAC-modified gold surface. Polymerization was initiated by raising the temperature to 70 ◦ C on a hot plate and continued for 5 min (Scheme 1). After the polymerization process, the sensor was washed with DI water at room temperature for 1 h, in order to remove sacrificial PVA and to expose the macropores created in the polymeric structure. To assess the effects of macroporosity and the amino acid moieties in the polymeric structure, three different combinations were examined by varying the presence/absence of amino acid-based monomer (MAH) and, for control purposes, the sacrificial polymer (PVA): (i) amino acid functionality + macroporosity; (ii) amino acid functionality; (iii) macroporosity. The first combination had all functionalities, the second one had only histidine groups in a non-porous polymeric block, and the third one only had a basic macroporous structure. 2.3.3. Synthesis of porous polymeric film on QCM electrodes QCM measurements were used to monitor the Cu(II)-mediated supramolecular assembling of biomolecules in real time. QCM Au electrodes were coated with polymeric film having amino acid functionality and porous structure, following the protocol described in Section 2.3.2 (Scheme 1). 2.4. Selectivity and reversibility In order to show the selectivity and reversibility of the formation of supramolecular assembly, three different sets of experiments were performed by applying impedance measurements as mentioned before. Herein, bias potential, frequency range, amplitude, redox probe in the solution and its concentration were adjusted as 0.2 V, 0.1 Hz–0.1 MHz, 5 mV, and 10 mM [Fe(CN)6 ]3−/4− in 0.1 M KCl(aq), respectively. In the first set, metal ion coordination was performed with three different ions [Cu(II), Zn(II), and Pb(II)] at four different concentrations. In the second set, the orientation of four different biomolecules (albumin, immunoglobulin, transferrin, and
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Scheme 1. Schematic showing: (a) synthesis of functional monomer, and (b) creation of porous polymeric network. –1- Self-assembling of cysteine-based monomer and introduction of monomer solution, –2- thermal polymerization to create a porous structure with imidazole (histidine) functionalities, –3- Cu(II) chelation onto imidazole ring, and –4- human immunoglobulin orientation to form supramolecular assembly. The imidazole ring is mainly responsible for chelating Cu(II) ions while water molecules complete the coordination vacancies of Cu(II) ions, which are replaced with protein molecules during the supramolecular assembly formation.
anti-transferrin antibody) on the polymeric films was evaluated before and after Cu(II) ions coordination. Herein, the formation of supra-molecular assemblies between immunoglobulin/transferrin and anti-transferrin/transferrin pairs is also evaluated. In the third set, the elution/destruction of the supramolecular assemblies was investigated by using sodium chloride (NaCl) and ethylenediamine tetra acetic acid (EDTA) as eluting agents. NaCl was chosen to destroy the interaction between Cu(II) ions and the biomolecules and Na4 EDTA was chosen to destroy the interaction between Cu(II) ions and the polymeric films directly.
2.5. Instrumentation All voltammetry measurements were carried out using an Ivium Stat.XR electrochemical analyzer (Ivium Tech., Eindoven, Netherlands). A three-electrode cell with gold coated silica substrate as a working electrode, platinum wire as an auxiliary and an Ag/AgCl (3M KCl) reference electrode were used for the voltammetric measurements. A Q-Sense QCM-D Technology (Biolin Scientific Holding AB, Stockholm, Sweden) device was used for the QCM measurements. 5 MHz, 14 mm diameter, polished, ATcut, gold electrodes were used in these experiments. Atomic force microscopy (AFM) observations were carried out using ambient AFM (Nanomagnetics Instruments, Oxford, UK) in tapping mode in an air atmosphere. The AFM experimental parameters were: oscillation frequency (341.30 Hz), vibration amplitude (1VRMS ) and free vibration amplitude (2VRMS ). Samples were scanned at a 1 m/s scanning rate and 256 × 256 pixels resolution to obtain a view of a 2 m × 2 m area. Scanning electron microscopy (SEM) observations were performed using a LEO 155 Gemini (Zeiss, OR, USA). FTIR-ATR measurements were carried out using a Bruker Vertex 70 FTIR spectrometer (PerkinElmer, Waltham, MA, USA).
3. Results and discussion 3.1. Preparation and characterization of polymeric film on gold surfaces The morphology of the polymeric films (supported on the gold surface) was characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The presence of the functional groups in the polymeric films was confirmed using a FTIR spectrometer. The electrochemical behavior of the polymeric films was systematically investigated using differential pulse voltammetry (DPV). Fig. 1a–c shows the SEM images of polymeric films produced in the presence (Fig. 1a,b) and the absence (Fig. 1c) of PVA. As can be seen from Fig. 1a–b, the presence of PVA as a sacrificial polymer resulted in structures with different porosity, as expected. Washing the polymeric film with DI after polymerization exposed large pores of various sizes, which were randomly distributed in the body of the polymer. It has previously been reported that the distribution and size of the pores can be controlled by varying the concentration of sacrificial polymers (i.e. PVA, polyvinylpyridine) to adjust the pore size during copolymerization [23,24] but this was not explored here, since we focused on enhanced amino acid functionality rather than tuning the pore size. The absence of PVA caused relatively non-porous structures (Fig. 1c) (extra SEM images at different magnification were given in Supporting Information file, Figs. S1–S3). These findings were confirmed by the AFM images (Fig. 2a,b). As can be seen in the figure, the presence of PVA directly affected the surface morphology and porosity, while the resulting pores were from nano- to microscale. In order to confirm the presence and efficient removal of PVA from the polymeric network, as well as the chemical structure of the polymer, FTIR-ATR
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Fig. 1. SEM images of polymeric films: (a) with porous structure having amino acid functionality, (b) porous structure without amino acid functionality, and (c) only amino acid functionality.
Fig. 2. AFM images of polymeric films with porous structure having: (a) amino acid functionality, and (b) non-porous structure with amino acid functionality.
measurements were performed (Fig. 3). The bands around frequencies 1617 (C O, amide I) and 1470 cm−1 (N H bending, amide II) stemmed from the functional monomer, MAH and indicated its successful incorporation into the polymeric structure. After washing the polymer with DI, the dominant IR bands at 1554 and 1410 cm−1 arising from the carboxylate (87–90% hydrolyzed) and methylene ( CH2 ) groups of PVA disappeared. Also, the intensity of the band at 3310 cm−1 for alcohol ( OH) groups decreased after washing.
These results were consistent with the presence of PVA in the polymeric structure as a sacrificial polymer via physical adsorption and its successful removal after an efficient washing step [25]. The synthesis of a monomer with amino acid functionality and its incorporation into the polymeric structure is one, and probably the easiest, of the possible ways to integrate the biorecognition mechanism of biomolecules into biomimicking artificial networks. It is also well known that amino acid residues are the biologi-
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Fig. 3. FTIR-ATR spectra of the functional polymer (with porous structure having amino acid functionality) before and after removal of PVA.
cal building blocks for the selective substrate binding in a diverse range of biomolecules, such as proteins, enzymes etc. Therefore, we aimed to synthesize a polymerizable amino acid (l-histidine) derivative (named MAH) through a benzotriazole-mediated reaction (Scheme 1).
Differential pulse voltammetry (DPV) was used to evaluate the presence of histidine molecules in the polymeric film and their ability to coordinate Cu(II) ions due to the Lewis base character of the nitrogen atoms in the hetero-aromatic ring (imidazole moieties). The first step of the investigation was to study the effects of pH and incubation time on the chelation between the Cu(II) ions and the imidazole ring [26,27]. The pH values were varied by using different buffer systems in the pH range 4.0–7.4. We determined the optimal pH for Cu(II) ion chelation as 5.0 (Fig. 4a), where the nitrogen atoms have unpaired electrons in the imidazole ring and therefore are much more accessible for efficient Cu(II) ion chelation. At more alkaline pH values (pH > 7.0), a significant reduction in the electrochemical response was recorded; this could be due to the formation of the metal-oxide (CuO, Cu2 O etc) [28] and a subsequent reduction in capture efficiency. The chelation reaction rapidly reached a plateau value after about 120 s (Fig. 4b). After 120 s, the change in current peak was no longer significant and so 120 s was used as incubation time for further measurements. The results indicated that the adsorption kinetics of the polymeric film having both amino acid functionality and porous structure were quite fast and efficient. In order to understand the effect of the amino acid functionalities and porosity on the Cu(II) ion chelation, we synthesized three different polymers and compared their performance: (i) with both amino acid functionality and pores (PF1); (ii) with only amino acid functionality (PF2); and (iii) with only pores/without amino acid functionalities (PF3). Calibration curves were recorded with the different polymeric films over a Cu(II) ion concentration range of 0–1000 nM (Fig. 4d). As can be seen in Fig. 4c, the increase in Cu(II) ion concentration resulted in
Fig. 4. Differential pulse voltammetry responses of polymeric film having amino acid functionality and porous structure with: (a) various pH at 10 mV/s scan rate vs Ag/AgCl reference electrode, (b) effect of incubation time in acetate buffer pH: 5.0 (10 mM) at 10 mV/s scan rate vs Ag/AgCl refer-ence electrode, (c) analytical response of the modified electrode with increasing concentration of Cu, and (d) calibration curve.
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Fig. 5. Differential pulse voltammetry responses of three electrodes (PF1, with both amino acid functionality and pores; PF2, with only amino acid functionality; and PF3, with only pores/without amino acid functionalities) in acetate buffer (pH: 5.0, 10 mM) at 10 mV/s scan rate vs Ag/AgCl reference electrode.
an increase in the current peak for PF1. Due to the presence of amino acid functionality, PF2 also responded to the increasing concentration of Cu(II) ions, but the responsiveness of PF2 was much lower than that of PF1(see also Supporting Information Fig. S4). This result indicated that not only amino acid functionality, but also the film porosity has a synergic effect on controlling the recognition ability of the polymeric films. On the other hand, the polymeric film having only porous structure, showed small responses with respect to the presence of increasing concentration of Cu(II) (Fig. 5). This slight response could be due to the physical/chemical adsorption of the metal ions into the polymeric network (see also Supporting Information Fig. S5). As mentioned before, these results emphasized successful incorporation of the functional monomer, MAH into the backbone poly(HEMA), while imparting a macroporous structure to the polymeric film. 3.2. Supramolecular self-assembly on a QCM electrode surface
Fig. 6. Cu(II) ions captured on the gold surface of a QCM electrode coated with polymeric film having amino acid functionality and porous structure.
The main goal of this study was the integration of the biorecognition ability of the amino acid onto an easy-to-use sensing platform. On the basis of the results obtained for characterization and electrochemical evaluation reported above, we applied the same procedure to create functional polymeric films on the gold-coated surface of QCM crystals and to utilize their ability to form selfassembled supramolecular structures. Two different approaches were adopted: (i) time course real-time Cu(II) immobilization; and (ii) Cu(II)-mediated immunoglobulin G (IgG) orientation on the QCM chip. For the first, IgG was chosen as a model protein to optimize the parameters and compare the efficiencies of PF1 and PF2 films. In the second approach, we aimed to show how this approach might be utilized for forming a supramolecular assembly as a biosensor platform. The effect of Cu(II) ion concentration over the range 0.1–10 mg/mL, in acetate buffer (pH: 5.0, 10 mM) at constant flow rate 10 L/min, was evaluated (Fig. 6). The frequency shifts of the QCM were monitored and calculated using QTools software (Stockholm, Sweden). The QCM chip was exposed to Cu(II) ion solution at four different concentrations, followed by desorption and regeneration achieved using ethylenediamine tetraacetic acid (EDTA, 25 mM) solution and acetate buffer (pH 5.0), respectively. Fig. 7 illustrates that all steps, including equilibration–adsorptiondesorption-regeneration, were rapidly and reversibly completed in about 30 min. PF1 film on the QCM chip showed a linear
response over this concentration range with a very high regression coefficient (R2 ) of 0.9911. These results indicated the successful integration of this approach with the QCM while retaining the recognition abilities. The next step was to demonstrate the efficiency of Cu(II) mediated IgG recognition/orientation, which is a cornerstone of our main goal to develop supramolecular assemblies for biosensing platforms. For this purpose, we interacted IgG solutions over a concentration range of 0.5 and 100 g/mL (phosphate buffer, pH: 7.4, 10 mM) with a PF1-modified QCM chip with and without the Cu(II) ion chelation step, at a constant flowrate of 10 L/min (Fig. 7). Fig. 7a shows that the PF1 QCM chip responded to the increase in IgG concentration with a regression coefficient (R2 ) of 0.8657 (with a linear response of 86.6%) due to the specific interaction between histidine and IgG molecules [29,30]. However, the frequency shift and linearity improved with respect to regression coefficient (R2 : 0.9056 with a linear response of 90.56%) after Cu(II) chelation and delta frequency values decreased from 5.21 to 84.16 at an IgG concentration of 100 g/mL (Fig. 7b). The Cu(II) ion-mediated IgG orientation significantly affected the amount of IgG molecules immobilized (Fig. 7c), because of the specific interaction between Cu(II) ions and IgG which occurs through the Fc-fragment [31]. We should also mention here that all steps, including the equilibration-adsorption (six different concen-
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phate buffer (pH: 7.4, 10 mM), which indicated good reusability and cost-efficiency of the supramolecular assemblies. 3.3. Selectivity and reversibility As mentioned above, three different sets of impedance measurement experiments were performed (see Supporting Information Fig. S6 for EES circuit used). In order to allow for the variations in response of polymeric films due to film structure, metal ions and biomolecules, we converted the data into the normalized responses which were calculated as follows: R/R o = (R f − R o )/R o
Fig. 7. Supramolecular self-assembling on the gold surface QCM electrode coated with polymeric film having amino acid functionality and porous structure: (a) IgG assembling in absence of Cu(II) ions, (b) IgG assembling mediated via Cu(II) ions, (c) comparison of two systems.
trations) and desorption–regeneration were completed in about 100 min, which meant that each concentration reached equilibrium in about only 12 min. Also, we observed successful regeneration of the QCM chip under mild conditions by using 1 M NaCl in phos-
(A.1)
where Rf and Ro were the responses of electrodes after and before treatment with each analyte, respectively. As seen in Fig. 8a, Cu(II) ion coordination enhanced the biomolecule orientation on the polymeric films, especially in the cases of IgG and transferrin. This indicated an increase in affinity of biomolecules to adsorb onto surface (see also Supporting Information Figs. S7 and S8). The electrodes investigated, PF2 resulted in the highest responses for all biomolecules before and after Cu(II) ion coordination, due to the non-porous structure providing greater insulation, and hence higher resistivity. The transferrin orientation on Cu(II) ions showed the highest increase in responses because of its metal binding pocket. The increases were 8.17-, 5.91-, and 1.60- folds for PF1, PF2, and PF3 electrodes, respectively. These results also confirmed both successful Cu(II) ion coordination and the porous structure of the polymeric films as well as the incorporation of metal chelating groups (histidine in MAH monomer) into the polymeric backbone. Note that only PF1 included the porous structure and MAH groups. In addition to biomolecule orientation on Cu(II) ions, the formation of supramolecular assemblies between the recognizing biomolecules (IgG molecule and anti-transferrin antibody) and the target molecule (transferrin) was also monitored both in the presence and absence of Cu(II) ions (Fig. 8b). In this respect, we aimed to investigate how Cu(II) plays a major role during the supramolecular assembly formation process (see also Supporting Information Figs. S9 and S10). In the absence of Cu(II) ions, the responses were inhibited by addition of a second biomolecule (transferrin), which indicated competition and replacement of transferrin with the other biomolecules. Therefore, the responses were lower than those for both of recognizing biomolecules and transferrin. In the case of Cu(II) ion coordination, the interaction between the recognizing biomolecule and Cu(II) ions was protected and so the formation of supramolecular assembly was possible. The increases in the responses for IgG/transferrin pair were, in presence of Cu(II) ions, calculated as 3.41-, 3.92-, and 1.70-fold, whereas that for the anti-transferrin/transferrin pair were calculated as 7.50-, 3.41-, and 5.11-fold for PF1, PF2, and PF3 electrodes, respectively. The highest increase exhibited by PF1 indicated the high affinity of the electrodes resulting from the porous structure and histidine functionality as well as confirming the formation of a supramolecular assembly (see also Supporting Information Fig. S11 and S12). Fig. 8c showed the effects of the type and concentration of metal ions, which was another set of experiments to confirm ion coordination depending on the structural (porous vs. non-porous) and chemical (having histidine or not) features of the polymeric film. As seen in the figure, PF1 showed the most dependency on the type of metal ions and the least dependency on their concentration. These results indicated that: (i) metal ions were coordinated by histidine groups, (ii) metal ion coordination decreased permeability of the probe couple by blocking the pores in the polymeric structure, and (iii) the increase in the metal ion concentration did not effect on the response due to occupation of histidine functionalities. The non-porous structure of PF2 decreased the sensitivity of
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Fig. 8. The selectivity and reversibility of the formation of supramolecular assembly. (a) the effect of Cu(II) ion coordination on the selectivity of orientation of four different biomolecules, (b) the effects of Cu(II) coordination and the recognizing biomolecule on the formation of supramolecular assembly, (c) the effects of the type and concentration of metal ions, and (d) the reversibility of interactions between Cu(II) ions/biomolecules and Cu(II) ions/polymeric film. Herein, bias potential, frequency range, amplitude, redox probe in the solution and its concentration were adjusted as 0.2 V, 0.1 Hz–0.1 MHz, 5 mV, and 10 mM [Fe(CN)6 ]3−/4− in 0.1 M KCl(aq), respectively.
the polymeric film to the type of metal ions. However, the increase in the concentration caused significant variation in the responses due to the coordination of metal ions by histidine functionalities on the surface. In the case of PF3, both of the type and concentration of metal ions caused a considerable variation in the responses. In the third set of experiments, we aimed to show the reversibility of the formation of the supramolecular assembly; so the selective elution of only biomolecules (protecting Cu(II) coordination) and Cu(II) ions (the complete destruction of assembly) was evaluated by using NaCl and EDTA as the eluting agents (Fig. 8d). As seen in the figure, NaCl treatment helped to remove biomolecular assembly between anti-transferrin and transferrin while protecting the Cu(II) coordination on the PF1 film (see also impedance response of Cu(II) ion coordination in Supporting Information, Fig. S13). However, EDTA treatment resulted in a response the same as the plain PF1 electrode, which indicated a direct destruction of the supramolecular assembly through chelating of metal ions by means of EDTA molecules. We should mention that each step (NaCl and EDTA treatments) was performed separately after the formation of the supra molecular assembly. The results confirmed the selectivity and reversibility of the formation of supramolecular assemblies while illustrating the considerable potential of the materials developed for the easy design of new analytical biosensors.
4. Conclusions Biological elements such as amino acids, nucleotides, and monosaccharides, play a major role in biorecognition of biological molecules. Sometimes, a single change in sequence results in completely different biorecognition abilities. Histidine especially, as one of the more active functional amino acids, has a role in metal ion coordination and intermolecular communications. Inspired by this knowledge, we focused our attention on developing a biosensing platform through simple, efficient and selective supramolecular assembly. We synthesized polymerizable histidine derivatives to directly insert them into a porous polymeric network and to integrate their biorecognition capabilities into artificial surfaces. Histidine incorporation into the polymeric structure allowed faster recognition of Cu(II) ions (only 120 s) over a wide concentration range of 0–1000 nM. In addition, this enhanced the Cu(II)-ion mediated IgG recognition/orientation process and resulted in significant improvement in the linear regression coefficients obtained. We also demonstrated how this strategy could be used to construct supramolecular assemblies as recognition elements in biosensing platforms. The successful QCM results revealed that this simple approach should be applicable to a variety of sensing platforms. Also, the basic idea of metal ion-mediated supramolecular assembly formation, is very useful for developing an efficient biohybrid sensing interface with an improved response time and signal inten-
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sity as well as better linearity, which increased from a correlation coefficient of 0.8657–0.9056 as a consequence of Cu(II) ion chelation with histidine groups. In conclusion, the results demonstrate that this approach is simple, easy-to-use, selective, reversible, and reusable and should be applicable to different sensing platforms as an inexpensive way to develop efficient biohybrid sensing platforms. Acknowledgements The authors wish to acknowledge the Swedish Research Council (VR-2011-6058357) for generous financial support for this research. L. Uzun thanks to the European Commission for Marie Curie fellowship (MIFs4BioMed) with grant agreement number: 629251. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.05.030. References [1] C. Sanchez, H. Arribart, M. Madeleine, G. Guille, Biomimetism and bioinspiration as tools for the design of innovative materials and systems, Nat. Mater. 4 (2005) 277–288. [2] G.M. Whitesides, Bioinspiration: something for everyone, Interface Focus 5 (2015) 1–10. [3] M. Hussain, J. Wackerlig, P.A. Lieberzeit, Biomimetic strategies for sensing biological species, Biosensors 3 (2013) 89–107. [4] R. Vaidhyanathan, D. Bradshaw, J. Rebilly, J.P. Barrio, J.A. Gould, N.G. Berry, M.J. Rosseinsky, A family of nanoporous materials based on an amino acid backbone, Angew. Chem. Int. Ed. 45 (2006) 6495–6499. [5] B. Alberts, A. Johnson, J. Lewis, M. Raft, K. Roberts, P. Walter, Molecular Biology of the Cell, fourth ed., Garland Science, New York, USA, 2002. [6] A.N. Jha, S. Vishveshwara, J.R. Banavar, Amino acid interaction preferences in proteins, Protein Sci. 19 (2010) 603–616. [7] Y. Shimazaki, M. Takani, O. Yamauchi, Metal complexes of amino acids and amino acid side chain groups. Structures and properties, Dalton Trans. 38 (2009) 7854–7869. [8] S.H. Laurie, Comprehensive Coordination Chemistry, Pergamon Press, Oxford, 1987. [9] S.H. Laurie, Handbook of Metal–Ligand Interactions in Biological Fluids: Bioinorganic Chemistry, Marcel Dekker, New York, USA, 1995. [10] T. Kiss, Biocoordination Chemistry: Coordination Equilibria in Biologically Active Systems, Ellis Horwood, Chichester, UK, 1990. [11] E. Chow, J.J. Gooding, Peptide modified electrodes as electrochemical metal ion sensors, Electroanal 18 (2006) 1437–1448. [12] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Metal chelate affinity chromatography, a new approach to protein fractionation, Nature 258 (1975) 598–599. [13] E.B. Altintas, N. Tuzmen, L. Uzun, A. Denizli, Immobilized metal affinity adsorption for antibody depletion from human serum with monosize beads, Ind. Eng. Chem. Res. 46 (2007) 7802–7810. [14] A.A. Ozcan, R. Say, A. Denizli, A. Ersoz, L-Histidine imprinted synthetic receptor for biochromatography applications, Anal. Chem. 78 (2006) 7253–7258. [15] E. Rosini, G. Molla, C. Rossetti, M.S. Pilone, L. Pollegioni, S. Sacchi, A biosensor for all d-amino acids using evolved d-amino acid oxidase, J. Biotechnol. 135 (2008) 377–384. [16] G.J. Zhang, M.J. Huang, J.J. Ang, E.T. Liu, K.V. Desai, Self-assembled monolayer-assisted silicon nanowire biosensor for detection of protein–DNA interactions in nuclear extracts from breast cancer cell, Biosens. Bioelectron. 26 (2011) 3233. [17] J. Luo, Q.D. Zhang, Y.Y. Huang, G.Q. Liu, R. Zhao, Quartz crystal microbalance biosensor for recombinant human interferon- detection based on antisense peptide approach, Anal. Chim. Acta. 590 (2007) 91–97. [18] J. Zhang, H.L. Qi, Y. Li, J. Yang, Q. Gao, C.X. Zhang, Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex, Anal. Chem. 80 (2008) 2888–2894. [19] Q. Jiang, Y. Shi, Q. Zhang, N. Li, P. Zhan, L. Song, L. Dai, J. Tian, Y. Du, Z. Cheng, B. Ding, A self-assembled DNA origami-gold nanorod complex for cancer theranostics, Small 38 (2015) 5134–5141.
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Biographies Erdogan Ozgur received BS degree from department of chemistry education (2008) and MSc and PhD degrees in chemistry from Hacettepe University, Turkey (2011 and 2016). His research interests are molecular imprinted polymeric micro/nano materials, and their applications. Onur Parlak received his PhD degree in Bioelectronics from Linköping University, Biosensors and Bioelectronics Centre in September 2015. Earlier, he was visiting research intern at Nanyang Technological University, Materials Science and Engineering Department, Singapore in 2011. He received his master and bachelor degree from Izmir Institute of Technology, Department of Chemistry in 2011 and 2009, respectively. He was recently awarded by one of the most prestigious grant in Sweden, Knut Allice Wallenberg Foundation for postdoctoral research fellow at Stanford University, Materials Science and Engineering, working with Prof. Alberto Salleo, currently. Valerio Beni works with development of electrochemical chemo/bio-sensors, for environmental, clinical and food analysis, and their integration with hybrid-printed platform. Previously, Beni has worked as a Postdoctoral Researcher at the Irish Tyndall National Institute, a Marie Curie Fellow at the Universitat Rovira I Virgili in Spain and as an Assistant Professor at Linköping University. Beni holds a Ph.D. in Chemistry. His core expertise lies in the area of electrochemical chemo/bio-sensors and their applications in clinical, food and environmental analysis. Anthony P.F. Turner is currently Head of Division FM-Linköping University’s new Centre for Biosensors and Bioelectronics. His previous thirty-five year academic career in the UK culminated in the positions of Principal (Rector) of Cranfield University and Distinguished Professor of Biotechnology. Professor Turner has more than 600 publications and patents in the field of biosensors and biomimetic sensors and is best known for his role in the development of glucose sensors for home-use by people with diabetes. He published the first textbook on biosensors in 1987 and is Editor-In-Chief of the principal journal in his field, Biosensors & Bioelectronics, which he co-founded in 1985. Lokman Uzun is an Associate Professor at the Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey where he also received his PhD in 2008. He is the author of more than 100 articles in peer-review journals with a h-index value of 27 and one of Associate Editors of Advanced Materials Letters. He recently awarded by an European Union Marie-Curie fellowship with the Biosensors and Bioelectronics Centre, Linköping University, Sweden for two years. His research interest is mainly in materials science, surface modification, affinity interaction, polymer science, especially molecularly imprinted polymers and their applications in biosensors, bioseparation, food safety, and the environmental sciences.