Evaluation of Functional Features of Immobilized

Advances in Biomedicine and Health Science

Evaluation of Functional Features of Immobilized Enzymes Using Electrochemical Label-Free Methods MONICA FLORESCU Faculty of Medicine Transilvania University of Brasov Str. N. Balcescu Nr. 56, 500019, Brasov ROMANIA [email protected] Abstract: - Biosensors are analytical tools, based on the combination of a biological recognition compound and a physical transducer (sensor). Employing biomolecules as analytical tools offer advantages compared with conventional methods due to of their simplicity, specificity, selectivity and quick response for real-time analysis. Immobilized biomolecules usually show lower activity towards specific biomolecular interactions compared with free ones, thus biosensors stability and analytical performance depends on the matrix and on the immobilization process used. In this work glucose oxidase enzyme was immobilized using layer-by-layer (LbL) method on mediated electrodes for biosensors development. Ultrathin multilayer films with high order on a molecular scale could be obtained where enzyme is immobilized using electrostatic interaction. Label-free methods like Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were used to characterize the structural and functional features of immobilized biomolecules on electrode surface using LbL method by comparison with cross-linking method.

Key-Words: - enzyme-based biosensors, layer-by-layer method, cyclic voltammetry, electrochemical impedance spectroscopy. immobilization of the enzyme into a stable layer. The cross-linking method with glutaraldehyde for enzyme-based biosensors development is well known and widely used [2]. Nowadays, layer-bylayer (LbL) assembly represents an interesting methodology due to its simplicity and versatility. Ultrathin multilayer films with high order on a molecular scale could be obtained. One modality of LbL nanofabrication of nanoscale film structures is using polyelectrolyte multilayers (PEM) formed by sequential deposition of oppositely charged macromolecules that bind to each other by electrostatic interactions.

1 Introduction Biosensors are analytical tools, based on the combination of a biological recognition compound and a physical transducer (sensor). A biosensor is employing biochemical molecular-recognition properties as the basis for a selective analysis which can be used for early diagnosis. Using this type of devices employing biomolecules as analytical tools offer advantages compared with conventional methods due to of their simplicity, specificity, selectivity and quick response for real-time analysis. [1]. Electrochemical methods are seen as complementary to the clasical techniques, and are especially attractive because they allow the possibility of creating inexpensive, easy to use and instrumentation for real-time analysis of biomarkers, such as biosensors. Electrochemical methods are label-free methods which can be used to characterize the structural and functional features of immobilized biomolecules on electrode surface. Immobilized biomolecules usually show lower activity towards specific biomolecular interactions compared with free ones. The biosensor stability and its analytical performance depend on both the immobilization process, and the matrix used for

ISBN: 978-1-61804-190-6

2 Problem Formulation In this work cyclic voltammetry (CV) and EIS were used to characterize the structural and functional features of immobilized glucose oxidase enzyme, GOX, (as anionic polyelectrolyte at pH 7) together with poly(ethyleneimine) (PEI) as cationic polyelectrolyte on mediated electrode surface using LbL method by comparison with cross-linking method.

62


entrapment in a polymer matrix or membrane [9 and references therein]. The LbL assembly process can be performed in aqueous solutions under conditions that minimize protein denaturation, allows for precise control of the composition and ﬁlm thickness.

2.1 EIS - Impedance Spectroscopy EIS principle is based on the changes of the interfacial properties of the electrode in the presence of reversible redox couple using impedance measurements. Electrochemically inert species can be measured by EIS in the presence of a redox agent, such as cobalt phthalocyanine (CoPC), which undergoes oxidation and reduction at the surface of the electrode, thus making possible label-free detection [3-5]. Immobilized molecule interacts with its target analytes and a complex is formed, an increase of interfacial electron-transfer resistance can be detected by EIS [6]. The impedance Z of a system is determined by the ratio of small amplitude applied voltage perturbation (V(t)) and current response (I(t)). AC impedance spectra for each electrode are recorded sequentially in the same measurement electrolyte, usually in a frequency range of 0.1 Hz to 100 kHz and AC amplitude constant. The impedance spectrum obtained by frequency scanning of impedance allows the surfaces, film and interface together with exchange and diffusion processes characterization. Therefore, an equivalent circuit consisting usually of resistances and capacitances is used for impedance spectrum analyzing. The circuit elements represent the different physicochemical properties of investigated system. Z is a complex value because I(t) vary both in amplitude and phase compared to V(t).

3 Problem Solution Glucose oxidase-based biosensor have been developed using screen-printed electrodes (SPEs) coated with cobalt phthalocyanine (CoPC) as electrochemical mediator, Layer-by-layer immobilization ultrathin multiple layers are physically adsorbed on top of working electrode. Easy to prepare and high bond strength appears where enzyme is immobilized using electrostatic interaction between negatively charged GOX and positively charged PEI.

2.1 Material and methods The enzyme glucose oxidase (GOX) from Aspergillus niger Type II, compatible glucose substrate, poly(ethyleneimine) solution (PEI), 50 % (w/v) in H2O, glutaraldehyde 25% (GA) and bovine serum albumin (BSA) were purchased from SigmaAldrich, Inc, USA. The supporting electrolyte was 0.1 M phosphate buffer (pH 7.0). GOX was dissolved at a concentration of 10 mg mL-1 in a pH 7.0 phosphate buffer containing 20 mg mL-1 BSA and 0.5 M NaCl. The concentration of PEI solution was 20 mg mL-1 in buffer. The concentration of GA solution used for cross-linking was 2.5% in water. All chemicals used for the preparation of stock and standard solutions were of analytical reagent grade and purchased from Sigma-Aldrich or Merck. All solutions were prepared with bi-distilled water. SPEs were prepared accordingly to procedures previously described [10] (gift from Prof. J.-L. Marty, University of Perpignan Via Domitia, France). The diameter of the surface area of the working electrode is 0.4 mm, the auxiliary electrode is a 16 x1.5 mm curve line surrounding on two sides the working electrode and the Ag/AgCl pseudoreference electrode is a 5 x1.5 mm straight line positioned on the third side of the working electrode.

2.2 Layer-by-layer immobilization method The matrix used for immobilization of the enzyme into a stable layer is an important element in biosensors development. This layer should keep closer as possible the enzyme at the electrode surface allowing a fast electron transfer. In the same time the enzymatic activity of immobilized GOX has to be preserved as much as possible. Therefore, their deeply embedded electron-transfer centers in the insulated peptide backbones, possible adsorptive denaturation of proteins, and/or unfavorable orientations of the enzyme at solid surfaces need to be avoided [7]. It have been demonstrated in the literature that carbon has been the substrate of choice for many enzyme immobilization studies, given the good compatibility between the enzyme layer and the electrode substrate [8]. The polyelectrolyte nanoscale film provides an ideal environment for retaining the enzyme activity and promoting the electron-transfer reactivity of GOX compared with covalent binding, carrier binding, cross-linking of enzyme, encapsulation and

ISBN: 978-1-61804-190-6

2.1.1 Electrochemical measurements Measurements were made in a one-compartment cell containing modified screen-printed electrodes.

63


when the first PEI/GOX bilayer is deposited on the CoPC-SPE surface. As the other bilayers are deposited on the electrode surface the voltammograms became wider and starting with the fourth bilayer the peak highness begins to stabilise. However, the assembly of the fifth bilayer does not further increase the peak current. The possible explanation for this phenomenon is that the distance between the fifth GOX layer and the electrode is great enough to diminish the electron communication. Fig. 1b demonstrates that the assembled GOX retains its catalysing activity. When glucose is added to the buffer solution the reduction peak of CoPC decreases along with the increase in the glucose concentration. The decrease of the reduction current can be employed to determine the glucose concentration and probe the enzyme activity of GOX in the films.

Voltammetric and EIS experiments were carried out using the PC-controlled Autolab PGSTAT Potenstiostat with FRA32M for Impedance analysis (Eco Chemie B.V., The Netherlands) controlled with GPES and FRA software. The CoPC mediator is applied on the surface of the electrode to shuttle the electrons between the redox centers of GOX and the electrode. All cyclic voltammograms were done by sweeping the electrode potential between -1.0 and 0.8 V at ambient temperature (25±2°C) in phosphate buffer solution, pH 7.0. For electrochemical impedance measurements a sinusoidal voltage perturbation of amplitude 10 mV rms was applied in the frequency range between 10 kHz and 0.1 Hz.

2.1.2 Enzyme immobilization Enzyme immobilization was performed by dropcoating procedure using 5 ul of appropriate solutions. Two immobilization methods onto CoPCmediated SPE were performed and compared. One method was carried out using LbL nanofabrications, where well-defined nanoscale film structures are built on surfaces by self-assembly. PEMs are formed by sequential deposition of oppositely charged macromolecules via alternate 20-min adsorption from the appropriate solutions: GOX and PEI that bind to each other by electrostatic interactions. There were deposited up to four different bilayers of PEI/GOX. A second immobilization cross-linking method using GA was used for comparison. For this purpose 5 µl containing a mixture of enzyme solution and GA with the concentrations presented in section 2.1 were dropped on SPE surface for 2 h. Both types of electrodes were allowed to dry for another 2 h at room temperature.

(a)

(b)

PBS PBS+1 mM gluc PBS+5 mM gluc

40

20 0 -20

20 0 -20 -40

-40 -0.8

-0.4

0.0

E (V)

0.4

0.8

-1.2

-0.8

-0.4

0.0

0.4

0.8

E/V

Fig. 1. CVs for: (a) CoPC-SPE in PBS and different number of PEI/GOX bilayers; (b) four PEI/GOX bilayers CoPC-SPE in two concetrations of glucose

2.2.2 EIS - Impedance Spectroscopy EIS was used to characterize the physical and interfacial properties of PEI/GOX bilayers film structure obtained by LbL nanofabrication. Spectra recorded CoPC-SPE electrodes, at 0 V applied potentials in 0.1M pH 7.0 phosphate buffer solutions are shown in Fig. 2. The form of the spectra for both types of electrode is similar at applied potential tested indicating a relatively high charge-transfer resistance and capacitance of the CoPC-SPE interface in buffer [9]. When the first PEI/GOX bilayer is deposited on the CoPC-SPE surface, with a dramatic decrease of both interface charge-transfer resistance and capacitance of the CoPC-SPE occur proving the adsorption of the PEI/GOX molecules on the electrode surface leading to the formation of a film (Fig. 2a). More PEI/GOX molecules are adsorbed more the thickness of the deposited substance increased and capacitance at the electrode-electrolyte interface decreases with surface coverage (Fig. 2b). Similar with CVs results

2.2 Results and discussion The CoPC-mediated biosensors responses were analyzed by CV and EIS in phosphate buffer pH 7.0. Effect of number of immobilized bilayers on LbL biosensor response was analyzed also by comparison with cross-linking biosensors.

2.2.1 Cyclic voltammetry Biosensors response was analyzed by recording the current when the electrode potential was swept between -1.0 and 0.8 V with scan rate of 0.05 Vs-1. As it can be seen from the Fig. 1a a shifted welldefined reduction peak of CoPC can be observed

ISBN: 978-1-61804-190-6

60

I/uA

I (µA)

40

-1.2

80

PBS PBS+ 1BL PBS+ 2BL PBS+ 3BL PBS+ 4BL

60

64


methods show that four PEI/GOX bilayers deposited on CoPC-SPE promotes easier electron-transfer reaction.

the assembly of the fifth bilayer does not further changes in impedance spectra. When glucose added the interface charge-transfer resistance of the CoPCSPE at buffer solution increases along with the increasing in the glucose concentration, being also in concordance with CV results. This fact suggests that charge transfer processes are controlling the surface phenomena. Difference in impedance spectra of CoPC coated with PEI/GOX (a)

E6_1P+1G E6_2P+2G E6_3P+3G E6_4P+4G

200

E6 E6_1P+1G

1600

References: [1] T.M. Canh, Biosensors, Chapman & Hall, London, 1993. [2] Betancourt et al., Glutaraldehyde in Protein Immobilization. A Versatile Reagent, Methods in Biotechnology, Vol. 22, 2006, pp. 57-64. [3] E. Komarova, et al., New multispecific array as a tool for electrochemical impedance spectroscopy-based biosensing, Biosensors and Bioelectronics, Vol. 25, 2010, pp. 1389–1394. [4] F. Lisdat, D. Schäfer, The use of electrochemical impedance spectroscopy for biosensing. Analytical and Bioanalytical Chemistry, Vol. 391, 2008, pp. 1555-1567. [5] A.H. Loo, A. Bonanni, M. Pumera, Impedimetric thrombin aptasensor based on chemically modified graphenes. Nanoscale, Vol. 4, 2012, pp. 143-147. [6] C.M.A. Brett, Electrochemical impedance spectroscopy for characterization of electrochemical sensors and biosensors, ECS Transactions, Vol. 13, 2008, pp. 67-80. [7] C. Fan, G. Li, D. Zhu, Recent progress in immobilized enzyme-based reagentless electrochemical biosensors, Current Topics in Analytical Chemistry, Vol. 3, 2002, pp. 233– 251. [8] S. Sotiropoulou, V. Gavalas, V. Vamvakaki, N.A. Chaniotakis, Novel Carbon Materials in Biosensor Systems, Biosensors and Bioelectronics, Vol. 18, 2003, pp. 211-215. [9] M. Florescu, C.M.A. Brett, Development and evaluation of electrochemical glucose enzyme biosensors based on carbon film electrodes Talanta, Vol. 65, 2005, pp. 306–312. [10] G. Valdes-Ramirez, et al., Sensitive amperometric biosensor for dichlorovos quantification: Application to detection of residues on apple skin, Talanta, Vol. 74, 2008, pp. 741- 746.

(b)

150

-Z''/kOhm

-Z''/kOhm

1200

800

100

50

400

0 0

400

800

1200

0

1600

0

50

Z'/kOhm

100

150

200

Z'/kOhm

(c)

60

(d)

50

40

-Z''/kOhm

-Z''/kOhm

40

20

E6_PBS E6_1mM gluc E6_5 mM gluc

30

20

E2_(SE+GA) E6_(4P+4G) 10

0

0 0

20

40

60

Z'/kOhm

0

10

20

30

40

50

Z'/kOhm

Fig. 2. Complex plane impedance plots recorded pH 7 buffer solutions for 0 V at: (a) CoPC-SPE and one PEI/GOX bilayer, (b) up to four PEI/GOX bilayers deposited, (c) in different concentration of glucose, (d) PEI/GOX bilayers and GA-GOX at CoPC f bilayers and GA-GOX can be observed in the Fig. 2d. The lower interface charge-transfer resistance for reaction products detection in the presence of glucose in buffer solution support the hypothesis that four PEI/GOX bilayers deposited on CoPC-SPE promotes easier electron-transfer reaction.

4 Conclusion Successfully immobilized multiple layers of glucose oxidase using LbL self-assembly were realized. The label-free methods like Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were used to characterize the structural and functional features of immobilized biomolecules (up to four bilayers of PEI/GOX) on electrode surface using LbL method by comparison with cross-linking method. This work opens future perspectives for development of label-free biosensors using layerby-layer modification strategy. More PEI/GOX molecules are adsorbed more the thickness of the deposited substance increased with surface coverage up to four PEI/GOX bilayers. Both electrochemical

ISBN: 978-1-61804-190-6

65