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Well-ordered thin films as practical components of biosensors
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Thin Solid Films 516 (2008) 1171 – 1174 www.elsevier.com/locate/tsf
Well-ordered thin films as practical components of biosensors Joanna Cabaj a , Krzysztof Idzik a , Jadwiga Sołoducho a,⁎, Antoni Chyla b , Jolanta Bryjak c , Jacek Doskocz a a b
Faculty of Medicinal Chemistry and Microbiology, Department of Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland Institute of Physical and Theoretical Chemistry, Department of Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland c Faculty of Bioorganic Chemistry, Department of Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland Available online 14 June 2007
Abstract A Langmuir–Blodgett (LB) film consisting of glutaraldehyde as the binding site for enzyme molecule, an amphiphilic N-alkyl-bis(thiophene) carbazole (BTC7) as the additional electron mediator, and 22-tricosenoic acid (TA) as the matrix for these molecules were deposited on a hydrophilic substrate. Laccase was then covalently immobilized on the film via glutaraldehyde (GA). The sensitivity of this sensor was about twice as high as that of laccase sensor prepared with the same LB film as above but without the bis(thiophene)carbazole derivative. The laccase LB film exhibited enzyme activity. © 2007 Elsevier B.V. All rights reserved. Keywords: Langmuir–Blodgett films; Laccase; Carbazole derivative; Covalent immobilization; Biosensor
1. Introduction The structure and properties of Langmuir monolayers and Langmuir–Blodgett films of long-chain amphiphile molecules have been studied extensively for the last decades. By combining the results of experimental and theoretical studies, it has been possible to generate a consensus view of the character of packing patterns in the several condensed phases that monolayers and films of these molecules exhibit. After the initial work on bioactive molecules in monolayers, it was obvious that researchers would attempt to transfer these solid films onto solid substrates. The Langmuir–Blodgett layers of amphiphile–proteins complexes with embedded immobilized enzymes are of great interest because of their biosensor applications [1]. Biological molecules, mainly enzymes, immobilized on transducers (such as amperometric or potentiometric electrodes or field effect transistors) are usually used as recognition elements [2]. Only very little enzymes or proteins can form sole LB films, but mostly protein molecules are incorporated to a solid surface by adsorption from solution and its binding to aliphatic acid film [3] via COOH group or by covalent crosslinking with glutaraldehyde (included in LB film). Moreover, in some cases,
⁎ Corresponding author. E-mail address: [email protected] (J. Sołoducho). 0040-6090/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2007.06.082
immobilization of laccase on solid substrate can improve its sensing stability and allow for their reuse. Sometimes, however, these methods of immobilization exhibit a drawback in that, that the surface concentration of enzyme molecules is hard to control [4]. Also conducting polymers or monomers have been extensively investigated as an electron mediators, which incorporated into LB films are responsible for enhancement of sensing effect of electrochemical sensor. For example, poly-3-dodecyl thiophene mixed with stearic acid can be an effective matrix for deposition of glucose oxidase, which retains its electroactivity and detects glucose [5]. Continuing our interest in chemistry of precursors of conducting and sensing materials [6], we present here the preliminary results of research of sensing properties of laccase from Cerrena unicolor covalently immobilized on LB film, including N-heptyl-bis(thiophene)carbazole (BTC7), which can be used for phenolic compounds detection. Laccase displays a broad specificity for the reducing substrates, catalyzing the oxidation of different phenols and aromatic diamines [4]. In our sensors, the function of enzyme immobilization was carried by glutaraldehyde added into the matrix film of 22tricosenoic acid (TA). The incorporated carbazole derivative was expected to facilitate the electron transfer, enhancing the sensing properties. Glutaraldehyde (GA) was spread on the water
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J. Cabaj et al. / Thin Solid Films 516 (2008) 1171–1174
Fig. 1. Computer generated models of N-heptyl-3,6-bis(thiophene)carbazole (BTC7) [13].
together with 22-tricosenoic acid and N-heptyl-bis(thiophene) carbazole as matrix molecule, and after compression on LB trough was deposited on hydrophilic quartz substrate. 2. Materials and methods 2.1. Materials Prior to deposition of LB films, the substrates were washed according to the standard way, following consecutive sonication in detergent (1% solution of DECON 90), rinsing with deionized water, etching in the ethanolic solution of KOH (Yirayama solution), to make them hydrophilic and finally copious rinsing with water and drying under the stream of dry air. An amphiphilic N-heptyl-bis(thiophene)carbazole (BTC7) (Fig. 1) was synthesized according to the procedure described by us earlier, using multi step method in which the main point was Stille coupling reaction [7]. Laccase (from Cerrena unicolor) was isolated and purified by standard method [8,9]. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonate) ABTS (Aldrich) was used as received. 2.2. Method The amphiphilic N-heptyl-bis(thiophene)carbazole, 22-tricosenoic acid and glutaraldehyde were dissolved in chloroform (Aldrich, HPLC grade) and mixed in equimolar proportions.
Concentration of each solution was maintained at ca. 1 mg ml− 1. About 50 μl of the mixture was spread on a water subphase (22 °C) and the monolayer was compressed with a movable barriers at 50 mm min− 1. The deposition was Y-type with a transfer ratio very close to unity, and the π–A isotherms were recorded by means of a commercial LB trough (KSV, System 5000), using a Pt hydrophilic Wilhelmy plate. Layers were transferred at withdrawing speed of 3.5 mm min− 1 and dipping speed of 15 mm min− 1, allowing 15 min of drying time between dipping cycles. The LB films were built up on hydrophilic quartz substrates (75 mm × 19 mm × 1.08 mm). The quality of LB films was confirmed by good surface pressure– area (π–A) isotherms and UV–VIS absorption spectra. For a covalent immobilization of laccase on modified surface an obtained LB film was sprinkled with 1 ml of aqueous laccase solution (1 mg ml− 1). In each case, immediately after applying the protein to thin LB layer, substrate was placed in desiccator. Process of immobilization was carried out for 24 h (2 h at room temperature and 22 h at 4 °C) and humidity around the substrate was maintained constant. Fig. 2 shows simple scheme for fabricating LB type film with immobilized proteins. According to previous experience, for determination of laccase activity the substrates were immersed in 25 ml of 2,2′-azino-bis(3ethylbenzthiazoline-6-sulphonate) ABTS (0.228 mmol l− 1, pH 5.25) [8] and incubated at 30 °C, under continuous solution stirring. The concentration of ABTS ensured optimum saturation of the enzyme with substrate (closely five as high as Km) and was successfully used in experiment. The fungal laccase oxidises ABTS to green-colored radical cation (ABTS •+) and colorimetric changes of ABTS solution can be spectroscopically measured. After washing off of the oxidised ABTS, the immobilized laccase was ready to react with fresh ABTS and the reaction could be repeated many times. The activity of the immobilized laccase Table 1 Compositions of Langmuir–Blodgett films and their transfer conditions Film
Fig. 2. Scheme for fabricating LB film as amphiphilic matrix for proteins immobilization.
a b
Composition BTC7
TA
GA
1
1 1
1 1
Pressure at transfer (mN m− 1)
Temperature of transfer (°C)
25 25
22 22
J. Cabaj et al. / Thin Solid Films 516 (2008) 1171–1174
Fig. 3. Surface pressure–area isotherms of amphiphilic BTC7 incorporated film a and BTC7 free film b, at 22 °C on pure water.
was monitored by continuous recording of the absorbance changes at 420 nm by means spectrometer Unicam Helios α. The specific activity was calculated from the measured absorbance value and the 1 Unit was defined as protein quantity, which in test conditions changed the absorbance by 0.0001 per minute. This specific activity related to the surface area of LB film (U cm− 2) expresses the activity of immobilized enzyme. Enzyme activity could be also expressed in relative units (%) whereas the 100% of the activity from the first absorbance changes could be taken. 3. Results and discussion Conditions and of the preparation and transfer of the LB films in this work and compositions of the films with (a) and without BTC7 (b) are listed in Table 1. The surface pressure–area (π–A) isotherms of films a and b on pure water, at 22 °C are presented in Fig. 3. The π–A curves
Fig. 4. Immobilized laccase activity during repeated reaction cycle, a) dependent on time, b) dependent on number of reaction cycle.
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Fig. 5. Activity of laccase immobilized on LB film, 1—with BTC7 as mediator, 2—without BTC7.
indicates that both a and b films are in solid state at high surface pressure. The surface per molecule area for film a is 33 Å2 whereas for film b it is 30 Å2. Film a collapses at surface pressure 52 mN m− 1 and film b at 48 mN m− 1. It is also seen that for mixed films (BTC7, TA) it is possible to obtain high surface pressures during compression and therefore tougher, more elastic films. In both cases tricosenoic acid, the model amphiphile, facilitates process of compression effectively. During compression BTC7 molecules are “pushed off” from the film, and may slip each under the other and form a “spoon like” structure (liquid condensed), as we reported earlier [7]. The glutaraldehyde molecules present in the film are additively mixed with aliphatic chains of amphiphiles, increasing the interchain volume and therefore increasing the average area per molecule. Since the immobilization of laccase on LB films was achieved through the crosslinking reaction with glutaraldehyde, its amount reflects the immobilized enzyme activity. In our case, the laccase incorporated to obtained LB film (1 × 10− 6 g) on quartz substrate had an initial enzyme activity of 30 U/cm2 (0.228 mmol l− 1ABTS, pH 5.25, 0.1 M citric acid– phosphate buffer). This value is about 0.1% of the activity of laccase in solution. The sensing activity of the laccase, covalently immobilized on LB films via glutaraldehyde, is rather stable and reproducible. Although, after the first 5 reaction cycles enzyme activity decreases to nearly 30% of the initial value but after that it keeps steady for at least 15 repeated reaction cycles (injection of 0.228 mM of ABTS) (Fig. 4). The temporal stability of the active enzyme is outstanding; it retains ca. 30% of its initial value.
Fig. 6. Scheme of mixed LB film with immobilized protein.
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J. Cabaj et al. / Thin Solid Films 516 (2008) 1171–1174
The important role of BTC7 molecules, acting as an electron mediator, is seen in Fig. 5. Comparing LB films, consisting (1) and not consisting (2) of BTC7 molecules, one can see that the enzyme activity in film 1 is about twice as high as in film 2. In general, a mediator could be a sort of ‘electron shuttle’ that, after being oxidised by the enzyme, diffuses away from the active site to oxidise any substrate that, for its size, could not enter the enzymatic pocket directly. In addition, the oxidised form of the mediator being structurally ‘diverse’ from the enzyme, might rely on a different mechanism of oxidation, thereby extending the range of substrates susceptible to the enzymatic action [10,11,12]. Suitable enzyme-mediator systems could also enable the environmentally benign. Understanding the role and mechanism of action of these mediators is a practical issue. ABTS is the most common mediator for laccase activity but not the most efficient one. As one could see from enzyme activity in Fig. 5, the presence of additional molecules with conjugated bonds (BTC7) in the system improves ABTS mediating efficiency significantly. Calculated from Fig. 3 mean area per molecule in mixed LB film suggests that BTC7 molecules are pushed off the carboxylic heads of TA layer (Fig. 6) with their aliphatic chains being parallel. It makes the occasion for glutaraldehyde (GA) to slip into the created channels (pockets) and interact, via aldehyde group with divalent sulphur of thiophene. In Fig. 6 we present model of laccase immobilized in LB film built of BTC7, TA, and GA as crosslinking activator. Ipso facto the other aldehyde groups of GA are exposed to react with lysine groups of the laccase. This “multilysinic” bonding of enzyme with LB film opens in some sense the access to its active centres. Moreover the grafting of enzyme onto LB layers can provide better electrosterical stabilisation, due to the high molecular weight of the protein and the numerous functional groups in the protein structure (carboxyl, amine, etc.) that are potential charge carriers [13,14]. 4. Conclusions A heterogeneous LB film, consisting of amphiphilic Nheptyl-bis(thiophene)carbazole (BTC7) and 22-tricosenoic acid modified by glutaraldehyde provides sites for successful laccase immobilization. This type of sensing system retains specific enzyme activity equal 0.1% of that of native laccase. Enzyme immobilized by this technique is active and stable for at least 15
reaction cycles (ABTS). Activity of protein deposited on LB films equipped with amphiphilic BTC7 was twice as high as that without BTC7 because of its electron mediating character. This fact of sensitization of sensing system with the presence of mediating conjugated amphiphile (BTC7) can be recognized as a successful step into enhancing biosensor activities, leading perhaps to manufacturing of different protein electrodes by use of conjugated, appropriate amphiphilic mediators. Sensing effects of biosensor construct with enzyme immobilized in LB film and interactions with mediated bis(thiophene) arylene derivatives are subject of our further investigations. Acknowledgments Authors gratefully acknowledge the financial support from the PBZ-KBN Grant No. 098/T09/2003/01. Authors would also like to thank Prof. Renata Bilewicz from Warsaw University and Prof. Jerzy Rogalski from Lublin University for sending laccase essential for researches. References [1] A. Pich, S. Bhattacharya, H.J.P. Adler, T. Wage, A. Taubenberger, Z. Li, K.H. van Pee, T. Bley, Macromol. Biosci. 301–310 (2006) 6. [2] D. Gidalevitz, Z. Huang, S.A. Rice, Biophys. J. 2797–2802 (1999) 76. [3] F. Davis, S.P.J. Higson, Biosens. Bioelectron. 1–20 (2005) 21. [4] D. Quan, W. Shin, Mater. Sci. Eng., C, Biomim. Mater., Sens. Syst. 113–115 (2004) 24. [5] T. Tatsuma, H. Tsuzuki, Y. Okawa, S. Yoshido, T. Watanabe, Thin Solid Films 145–150 (1991) 202. [6] J. Cabaj, J. Sołoducho, A. Nowakowska, A. Chyla, Electroanal. J. 801–806 (2006) 18 (8). [7] J. Cabaj, K. Idzik, J. Sołoducho, A. Chyla, Tetrahedron 758–764 (2006) 62. [8] J. Bryjak, Wiad. Chem. 691–746 (2004) 58. [9] J. Rogalski, A. Dawidowicz, E. Jóźwik, A. Leonowicz, J. Mol. Catal., B Enzym. 29–39 (1999) 6. [10] P. Baiocco, A.M. Barreca, M. Fabbrini, C. Galli, P. Gentili, Org. Biomol. Chem. 191–197 (2003) 1. [11] P. Brandi, A. D'Annibale, C. Galli, P. Gentili, A.S. Nunes Pontes, J. Mol. Catal., B Enzym. 61–69 (2006) 41. [12] D. Pal, P. Chakrabarti, J. Biomol. Struct. Dyn. 115–128 (2001) 19 (1). [13] J. Doskocz, M. Doskocz, S. Roszak, J. Sołoducho, J. Leszczynski, J. Phys. Chem., A 13989–13994 (2006) 110. [14] S. Yamaguchi, T. Shirasaka, S. Akiyama, K. Tamao, J. Am. Chem. Soc. 8816–88171 (2002) 24.
Thin Solid Films 516 (2008) 1171 – 1174 www.elsevier.com/locate/tsf
Well-ordered thin films as practical components of biosensors Joanna Cabaj a , Krzysztof Idzik a , Jadwiga Sołoducho a,⁎, Antoni Chyla b , Jolanta Bryjak c , Jacek Doskocz a a b
Faculty of Medicinal Chemistry and Microbiology, Department of Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland Institute of Physical and Theoretical Chemistry, Department of Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland c Faculty of Bioorganic Chemistry, Department of Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland Available online 14 June 2007
Abstract A Langmuir–Blodgett (LB) film consisting of glutaraldehyde as the binding site for enzyme molecule, an amphiphilic N-alkyl-bis(thiophene) carbazole (BTC7) as the additional electron mediator, and 22-tricosenoic acid (TA) as the matrix for these molecules were deposited on a hydrophilic substrate. Laccase was then covalently immobilized on the film via glutaraldehyde (GA). The sensitivity of this sensor was about twice as high as that of laccase sensor prepared with the same LB film as above but without the bis(thiophene)carbazole derivative. The laccase LB film exhibited enzyme activity. © 2007 Elsevier B.V. All rights reserved. Keywords: Langmuir–Blodgett films; Laccase; Carbazole derivative; Covalent immobilization; Biosensor
1. Introduction The structure and properties of Langmuir monolayers and Langmuir–Blodgett films of long-chain amphiphile molecules have been studied extensively for the last decades. By combining the results of experimental and theoretical studies, it has been possible to generate a consensus view of the character of packing patterns in the several condensed phases that monolayers and films of these molecules exhibit. After the initial work on bioactive molecules in monolayers, it was obvious that researchers would attempt to transfer these solid films onto solid substrates. The Langmuir–Blodgett layers of amphiphile–proteins complexes with embedded immobilized enzymes are of great interest because of their biosensor applications [1]. Biological molecules, mainly enzymes, immobilized on transducers (such as amperometric or potentiometric electrodes or field effect transistors) are usually used as recognition elements [2]. Only very little enzymes or proteins can form sole LB films, but mostly protein molecules are incorporated to a solid surface by adsorption from solution and its binding to aliphatic acid film [3] via COOH group or by covalent crosslinking with glutaraldehyde (included in LB film). Moreover, in some cases,
⁎ Corresponding author. E-mail address: [email protected] (J. Sołoducho). 0040-6090/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2007.06.082
immobilization of laccase on solid substrate can improve its sensing stability and allow for their reuse. Sometimes, however, these methods of immobilization exhibit a drawback in that, that the surface concentration of enzyme molecules is hard to control [4]. Also conducting polymers or monomers have been extensively investigated as an electron mediators, which incorporated into LB films are responsible for enhancement of sensing effect of electrochemical sensor. For example, poly-3-dodecyl thiophene mixed with stearic acid can be an effective matrix for deposition of glucose oxidase, which retains its electroactivity and detects glucose [5]. Continuing our interest in chemistry of precursors of conducting and sensing materials [6], we present here the preliminary results of research of sensing properties of laccase from Cerrena unicolor covalently immobilized on LB film, including N-heptyl-bis(thiophene)carbazole (BTC7), which can be used for phenolic compounds detection. Laccase displays a broad specificity for the reducing substrates, catalyzing the oxidation of different phenols and aromatic diamines [4]. In our sensors, the function of enzyme immobilization was carried by glutaraldehyde added into the matrix film of 22tricosenoic acid (TA). The incorporated carbazole derivative was expected to facilitate the electron transfer, enhancing the sensing properties. Glutaraldehyde (GA) was spread on the water
1172
J. Cabaj et al. / Thin Solid Films 516 (2008) 1171–1174
Fig. 1. Computer generated models of N-heptyl-3,6-bis(thiophene)carbazole (BTC7) [13].
together with 22-tricosenoic acid and N-heptyl-bis(thiophene) carbazole as matrix molecule, and after compression on LB trough was deposited on hydrophilic quartz substrate. 2. Materials and methods 2.1. Materials Prior to deposition of LB films, the substrates were washed according to the standard way, following consecutive sonication in detergent (1% solution of DECON 90), rinsing with deionized water, etching in the ethanolic solution of KOH (Yirayama solution), to make them hydrophilic and finally copious rinsing with water and drying under the stream of dry air. An amphiphilic N-heptyl-bis(thiophene)carbazole (BTC7) (Fig. 1) was synthesized according to the procedure described by us earlier, using multi step method in which the main point was Stille coupling reaction [7]. Laccase (from Cerrena unicolor) was isolated and purified by standard method [8,9]. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonate) ABTS (Aldrich) was used as received. 2.2. Method The amphiphilic N-heptyl-bis(thiophene)carbazole, 22-tricosenoic acid and glutaraldehyde were dissolved in chloroform (Aldrich, HPLC grade) and mixed in equimolar proportions.
Concentration of each solution was maintained at ca. 1 mg ml− 1. About 50 μl of the mixture was spread on a water subphase (22 °C) and the monolayer was compressed with a movable barriers at 50 mm min− 1. The deposition was Y-type with a transfer ratio very close to unity, and the π–A isotherms were recorded by means of a commercial LB trough (KSV, System 5000), using a Pt hydrophilic Wilhelmy plate. Layers were transferred at withdrawing speed of 3.5 mm min− 1 and dipping speed of 15 mm min− 1, allowing 15 min of drying time between dipping cycles. The LB films were built up on hydrophilic quartz substrates (75 mm × 19 mm × 1.08 mm). The quality of LB films was confirmed by good surface pressure– area (π–A) isotherms and UV–VIS absorption spectra. For a covalent immobilization of laccase on modified surface an obtained LB film was sprinkled with 1 ml of aqueous laccase solution (1 mg ml− 1). In each case, immediately after applying the protein to thin LB layer, substrate was placed in desiccator. Process of immobilization was carried out for 24 h (2 h at room temperature and 22 h at 4 °C) and humidity around the substrate was maintained constant. Fig. 2 shows simple scheme for fabricating LB type film with immobilized proteins. According to previous experience, for determination of laccase activity the substrates were immersed in 25 ml of 2,2′-azino-bis(3ethylbenzthiazoline-6-sulphonate) ABTS (0.228 mmol l− 1, pH 5.25) [8] and incubated at 30 °C, under continuous solution stirring. The concentration of ABTS ensured optimum saturation of the enzyme with substrate (closely five as high as Km) and was successfully used in experiment. The fungal laccase oxidises ABTS to green-colored radical cation (ABTS •+) and colorimetric changes of ABTS solution can be spectroscopically measured. After washing off of the oxidised ABTS, the immobilized laccase was ready to react with fresh ABTS and the reaction could be repeated many times. The activity of the immobilized laccase Table 1 Compositions of Langmuir–Blodgett films and their transfer conditions Film
Fig. 2. Scheme for fabricating LB film as amphiphilic matrix for proteins immobilization.
a b
Composition BTC7
TA
GA
1
1 1
1 1
Pressure at transfer (mN m− 1)
Temperature of transfer (°C)
25 25
22 22
J. Cabaj et al. / Thin Solid Films 516 (2008) 1171–1174
Fig. 3. Surface pressure–area isotherms of amphiphilic BTC7 incorporated film a and BTC7 free film b, at 22 °C on pure water.
was monitored by continuous recording of the absorbance changes at 420 nm by means spectrometer Unicam Helios α. The specific activity was calculated from the measured absorbance value and the 1 Unit was defined as protein quantity, which in test conditions changed the absorbance by 0.0001 per minute. This specific activity related to the surface area of LB film (U cm− 2) expresses the activity of immobilized enzyme. Enzyme activity could be also expressed in relative units (%) whereas the 100% of the activity from the first absorbance changes could be taken. 3. Results and discussion Conditions and of the preparation and transfer of the LB films in this work and compositions of the films with (a) and without BTC7 (b) are listed in Table 1. The surface pressure–area (π–A) isotherms of films a and b on pure water, at 22 °C are presented in Fig. 3. The π–A curves
Fig. 4. Immobilized laccase activity during repeated reaction cycle, a) dependent on time, b) dependent on number of reaction cycle.
1173
Fig. 5. Activity of laccase immobilized on LB film, 1—with BTC7 as mediator, 2—without BTC7.
indicates that both a and b films are in solid state at high surface pressure. The surface per molecule area for film a is 33 Å2 whereas for film b it is 30 Å2. Film a collapses at surface pressure 52 mN m− 1 and film b at 48 mN m− 1. It is also seen that for mixed films (BTC7, TA) it is possible to obtain high surface pressures during compression and therefore tougher, more elastic films. In both cases tricosenoic acid, the model amphiphile, facilitates process of compression effectively. During compression BTC7 molecules are “pushed off” from the film, and may slip each under the other and form a “spoon like” structure (liquid condensed), as we reported earlier [7]. The glutaraldehyde molecules present in the film are additively mixed with aliphatic chains of amphiphiles, increasing the interchain volume and therefore increasing the average area per molecule. Since the immobilization of laccase on LB films was achieved through the crosslinking reaction with glutaraldehyde, its amount reflects the immobilized enzyme activity. In our case, the laccase incorporated to obtained LB film (1 × 10− 6 g) on quartz substrate had an initial enzyme activity of 30 U/cm2 (0.228 mmol l− 1ABTS, pH 5.25, 0.1 M citric acid– phosphate buffer). This value is about 0.1% of the activity of laccase in solution. The sensing activity of the laccase, covalently immobilized on LB films via glutaraldehyde, is rather stable and reproducible. Although, after the first 5 reaction cycles enzyme activity decreases to nearly 30% of the initial value but after that it keeps steady for at least 15 repeated reaction cycles (injection of 0.228 mM of ABTS) (Fig. 4). The temporal stability of the active enzyme is outstanding; it retains ca. 30% of its initial value.
Fig. 6. Scheme of mixed LB film with immobilized protein.
1174
J. Cabaj et al. / Thin Solid Films 516 (2008) 1171–1174
The important role of BTC7 molecules, acting as an electron mediator, is seen in Fig. 5. Comparing LB films, consisting (1) and not consisting (2) of BTC7 molecules, one can see that the enzyme activity in film 1 is about twice as high as in film 2. In general, a mediator could be a sort of ‘electron shuttle’ that, after being oxidised by the enzyme, diffuses away from the active site to oxidise any substrate that, for its size, could not enter the enzymatic pocket directly. In addition, the oxidised form of the mediator being structurally ‘diverse’ from the enzyme, might rely on a different mechanism of oxidation, thereby extending the range of substrates susceptible to the enzymatic action [10,11,12]. Suitable enzyme-mediator systems could also enable the environmentally benign. Understanding the role and mechanism of action of these mediators is a practical issue. ABTS is the most common mediator for laccase activity but not the most efficient one. As one could see from enzyme activity in Fig. 5, the presence of additional molecules with conjugated bonds (BTC7) in the system improves ABTS mediating efficiency significantly. Calculated from Fig. 3 mean area per molecule in mixed LB film suggests that BTC7 molecules are pushed off the carboxylic heads of TA layer (Fig. 6) with their aliphatic chains being parallel. It makes the occasion for glutaraldehyde (GA) to slip into the created channels (pockets) and interact, via aldehyde group with divalent sulphur of thiophene. In Fig. 6 we present model of laccase immobilized in LB film built of BTC7, TA, and GA as crosslinking activator. Ipso facto the other aldehyde groups of GA are exposed to react with lysine groups of the laccase. This “multilysinic” bonding of enzyme with LB film opens in some sense the access to its active centres. Moreover the grafting of enzyme onto LB layers can provide better electrosterical stabilisation, due to the high molecular weight of the protein and the numerous functional groups in the protein structure (carboxyl, amine, etc.) that are potential charge carriers [13,14]. 4. Conclusions A heterogeneous LB film, consisting of amphiphilic Nheptyl-bis(thiophene)carbazole (BTC7) and 22-tricosenoic acid modified by glutaraldehyde provides sites for successful laccase immobilization. This type of sensing system retains specific enzyme activity equal 0.1% of that of native laccase. Enzyme immobilized by this technique is active and stable for at least 15
reaction cycles (ABTS). Activity of protein deposited on LB films equipped with amphiphilic BTC7 was twice as high as that without BTC7 because of its electron mediating character. This fact of sensitization of sensing system with the presence of mediating conjugated amphiphile (BTC7) can be recognized as a successful step into enhancing biosensor activities, leading perhaps to manufacturing of different protein electrodes by use of conjugated, appropriate amphiphilic mediators. Sensing effects of biosensor construct with enzyme immobilized in LB film and interactions with mediated bis(thiophene) arylene derivatives are subject of our further investigations. Acknowledgments Authors gratefully acknowledge the financial support from the PBZ-KBN Grant No. 098/T09/2003/01. Authors would also like to thank Prof. Renata Bilewicz from Warsaw University and Prof. Jerzy Rogalski from Lublin University for sending laccase essential for researches. References [1] A. Pich, S. Bhattacharya, H.J.P. Adler, T. Wage, A. Taubenberger, Z. Li, K.H. van Pee, T. Bley, Macromol. Biosci. 301–310 (2006) 6. [2] D. Gidalevitz, Z. Huang, S.A. Rice, Biophys. J. 2797–2802 (1999) 76. [3] F. Davis, S.P.J. Higson, Biosens. Bioelectron. 1–20 (2005) 21. [4] D. Quan, W. Shin, Mater. Sci. Eng., C, Biomim. Mater., Sens. Syst. 113–115 (2004) 24. [5] T. Tatsuma, H. Tsuzuki, Y. Okawa, S. Yoshido, T. Watanabe, Thin Solid Films 145–150 (1991) 202. [6] J. Cabaj, J. Sołoducho, A. Nowakowska, A. Chyla, Electroanal. J. 801–806 (2006) 18 (8). [7] J. Cabaj, K. Idzik, J. Sołoducho, A. Chyla, Tetrahedron 758–764 (2006) 62. [8] J. Bryjak, Wiad. Chem. 691–746 (2004) 58. [9] J. Rogalski, A. Dawidowicz, E. Jóźwik, A. Leonowicz, J. Mol. Catal., B Enzym. 29–39 (1999) 6. [10] P. Baiocco, A.M. Barreca, M. Fabbrini, C. Galli, P. Gentili, Org. Biomol. Chem. 191–197 (2003) 1. [11] P. Brandi, A. D'Annibale, C. Galli, P. Gentili, A.S. Nunes Pontes, J. Mol. Catal., B Enzym. 61–69 (2006) 41. [12] D. Pal, P. Chakrabarti, J. Biomol. Struct. Dyn. 115–128 (2001) 19 (1). [13] J. Doskocz, M. Doskocz, S. Roszak, J. Sołoducho, J. Leszczynski, J. Phys. Chem., A 13989–13994 (2006) 110. [14] S. Yamaguchi, T. Shirasaka, S. Akiyama, K. Tamao, J. Am. Chem. Soc. 8816–88171 (2002) 24.