Colloids and Surfaces B: Biointerfaces 116 (2014) 378–382
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Influence of immobilization protocol on the structure and function of surface bound proteins Alexej Kreider, Stephan Sell, Thomas Kowalik, Andreas Hartwig* , Ingo Grunwald Fraunhofer Institute for Manufacturing Technology and Advanced Materials, Wiener Strasse 12, Bremen 28359, Germany
a r t i c l e
i n f o
Article history: Received 6 March 2013 Received in revised form 8 July 2013 Accepted 8 July 2013 Keywords: Covalent immobilization Horseradish peroxidase PM-IRRAS Bioactivity Biofunctionalization
a b s t r a c t A new coupling strategy for biomacromolecules with (3-mercaptopropyl)trimethoxysilane (3MPTMS) and 11-(triethoxysilyl)undecanal (TESU) on gold surfaces is. This immobilization protocol was utilized for the enzyme horseradish peroxidase (HRP). To study the reactions and resulting structures, PM-IRRAS measurements were performed. PM-IRRAS shows there is structure preservation of the HRP when the new coupling strategy is used in contrast to non-specific adsorption on gold. The biological activity of adsorbed and immobilized HRP was measured by the enzyme catalyzed oxidation of 3,5,3 ,5 -tetramethylbenzidine. Covalent immobilization of HRP on TESU film compared to physisorption of HRP shows higher enzyme activity on gold surfaces, confirming the structural preservation detected by PM-IRRAS. c 2013 Elsevier B.V. All rights reserved.
1. Introduction Technological advancement in areas such as chemical sensors, biosensors, biochips [1,2], and coating materials [3,4] requires a chemical and physical understanding of surface processes. When biomacromolecules such as enzymes are immobilized on support materials, it is essential to conserve the biological activity and accessibility of the catalytically active sites of the enzymes [5–7]. In this context, the formation of an organic layer is the most widely used strategy for the covalent immobilization of biomolecules on surface materials using self-assembled monolayers [8,9]. The formation of organic monolayer films by self-assembly is a common approach for modification of a variety of metals using alkanethiols [10] and alkoxysilanes [11] with different functional groups. The groups are often carboxyl groups with the disadvantage of the need for activation with N-hydroxysuccinimide (NHS) ester and 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC) [12]. The aim of the work presented here is to introduce an alternative immobilization strategy, using the example of horseradish peroxidase (HRP). The thiosilane (3-mercaptopropyl)trimethoxysilane (3MPTMS) is used to functionalize a gold surface via the mercapto group. The hydrolyzable methoxysilyl groups of the surface immobilized 3MPTMS are used to graft the aldehyde terminated organosilane 11(triethoxysilyl)undecanal (TESU) via a sol–gel reaction on the surface
* Corresponding author. Tel.: +49 04212246470; fax: +49 04212246430. E-mail address: [email protected] (A. Hartwig).
c 2013 Elsevier B.V. All rights reserved. 0927-7765/$ - see front matter http://dx.doi.org/10.1016/j.colsurfb.2013.07.022
[13]. The resulting aldehyde modified surfaces were used to immobilize p-aminophenol as a model molecule or HRP as a biomacromolecule having a known secondary structure and an easily measurable bioactivity. The amine groups of lysine residues from the HRP can react with the surfaces’ aldehyde groups forming a Schiff’s base (imine) which is further reduced via reductive amination to a stable secondary amine using sodium cyanoborohydride NaCNBH3 [14,15]. Water, alcohols, and other oxygen nucleophiles typical for biological fluids do not compromise the yields of reductive amination [16,17]. Scheme 1 illustrates the overall strategy for surface immobilization described in this work. In order to analyze the adsorption phenomena and adhesion processes of biomolecules on the surfaces, a variety of measurement techniques can be used including infrared reflection absorption spectroscopy (IRRAS) [18], time of flight secondary ion mass spectrometry (ToF-SIMS) [19], X-ray photoelectron spectroscopy (XPS) [19], and atomic force microscopy (AFM) [20]. Of these measurement techniques, IRRAS is a particularly sensitive and nondestructive technique for acquiring information about, for example, the formation of chemical bonds with the substrate, the conformation, hydrogen-bonded structures, and the orientation of functional groups in the adsorbed substances on the metal surfaces [21]. IRRAS is only partially suitable for samples with an ultrathin film (thickness less than 10 nm) as the measurement time necessary to get an acceptable signal-tonoise ratio becomes several hours [22] and the long measurement time often leads to spectral artifacts due to small changes in the atmospheric composition, especially the water vapor content. For this reason, PM-IRRAS was developed [23]. The polarization modulation (PM) is a very efficient way of discriminating near-surface absorptions from the isotropic strong absorptions occurring in the sample environment [22]. Due to PM, the sample with thin film is also the
Alexej Kreider et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 378–382
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Scheme 1. Strategy for the immobilization of the biomolecule HRP and the model compound p-aminophenol on the gold surface (H&C = hydrolysis and condensation).
reference sample measured at the same time. Therefore, omnipresent contaminations such as hydrocarbons are also detected. They are often overlooked in conventional IRRAS experiments but their presence is, for example, known from XPS experiments. There are several works that use the enzyme HRP to determine the catalytic activity at interfaces [24–27]. In this study we have also chosen HRP as the model enzyme for the readily detectable oxidation of 3,5,3 5 -tetramethylbenzidine diamine to 3,5,3 5 tetramethylbenzidine diimine (Scheme 2). Josephy et al. [28] described the mechanism of oxidation of 3,5,3 ,5 -tetramethylbenzidine using HRP.
2.2. IR spectra ATR-IR spectra were recorded between 4000 and 650 cm−1 on a Bruker Equinox 55 FT-IR-spectrometer (Bruker Optik, Germany) equipped with Golden GateTM ATR unit A531G (Specac) with a resolution of 4 cm−1 and 32 scans. For the PM-IRRAS experiments a polarization modulation accessory (Bruker PMA-50) was used with MCT detector and a photoelastic modulator controller (PEM-90 controller) from Hinds Instruments (HINDS model II/Zn50 with a nominal frequency of 50 kHz, retardation range for half wave 1–10 μ m, and aperture 14 mm) coupled with a Bruker Tensor 27 FT-IR spectrometer. The spectra were an average of 45 min recording time and were taken at a spectral resolution of 4 cm−1 and an incident angle of 83.5◦ . 2.3. Immobilization protocol
2. Experimental 2.1. Materials Au/SiO2 wafers were obtained from Si-Mat Silicon Materials (diameter 100 mm, orientation P/Bor 1 0 0, thickness 525 ± 25 μ m, single polished). (3-Mercaptopropyl)trimethoxysilane (3MPTMS, 95%) and 11-(triethoxysilyl)undecanal (TESU, 90%) were purchased from ABCR (Karlsruhe, Germany). Horseradish peroxidase (HRP, Pcode: 101161191), triethylamine (TEA, 99.5%), methanol (≥99.9%), ethanol (96%), sodium hydrogen carbonate (≥99%, NaHCO3 ), sodium carbonate (≥99%, Na2 CO3 ), and sodium cyanoborohydride (5M, NaBH3 CN) were purchased from Sigma–Aldrich (Steinbach, Germany). One Step Ultra TMB-ELISA was acquired from Pierce Protein Research Products (ThermoScientific, Bonn, Germany). Water was obtained from an UHQ II ELGA water purification system (18 M/cm).
3MPTMS layers were formed at room temperature (RT) on the gold surface by immersion in a solution of 20 mM 3MPTMS in methanol for 2 h (Scheme 1). Then the silanized substrate was rinsed with methanol for 1 h at a shaking frequency of 100 min−1 using an orbital shaker and dried under a nitrogen stream. The samples were then dried in an exsiccator (200 mbar) for 16 h. The hydrolysis and condensation of the 3MPTMS on the substrates were carried out at 80 ◦ C by immersing the 3MPTMS-modified surfaces in 0.1 M HCl for 1 h, 2 h, and 3 h. Subsequently, the prehydrolyzed 3MPTMS layer was cleaned with double distilled water and dried in a nitrogen stream for about 1 min. The silanization process was carried out by incubating the prehydrolyzed 3MPTMS layer in a 96% ethanol solution containing 2% TESU and 1% TEA for 2 h at RT. Then the surfaces were rinsed with ethanol for 10 min at 100 min−1 using an orbital shaker and heated for 2 h at 110 ◦ C. The TEA acts as catalyst for the hydrolysis and condensation
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Scheme 2. Illustration of the applied enzymatic assay. (A) Oxidation of 3,5,3 ,5 -tetramethylbenzidine using HRP. (B) Covalent immobilization of HRP on the TESU-film. (C) Physical adsorption of HRP on the gold surface.
reaction of TESU with the 3MPTMS layer [29,30].
3. Results and discussion 3.1. Protocol for covalent protein immobilization
2.4. Coupling of p-aminophenol and horseradish peroxidase to the aldehyde functionalized gold surfaces The aldehyde-modified substrates for bonding p-aminophenol and the HRP by reductive amination [31,32] were placed in a 0.05 M carbonate buffer solution (pH 9.6) containing 168 mg/20 mL paminophenol or 2 mg/10 mL HRP for 6 h at RT. Then 800 μ L 5 M NaCNBH3 were added to the solutions to reduce the imine product formed on the surfaces. Reaction was for 3 h at RT. Subsequently, the substrates were rinsed once with carbonate buffer (pH 9.6) and once with double distilled water at 100 min−1 for 2 h using an orbital shaker.
2.5. Determination of the activity of immobilized HRP on the surface The activity of immobilized HRP on the TESU film or nonspecifically directly on the gold surface was determined by measuring the color development based on the 1-Step Ultra TMB-ELISA instruction. 3,3 ,5,5 -Tetramethylbenzidine (TMB) was used as the chromogen. To carry out the experiment one drop with a volume of 50 μ L of 1-Step Ultra TMB-ELISA was placed on the sample surface and incubated in the dark for 15 min at room temperature. The solution turned blue. The reaction was stopped by adding 50 μ L of 2 M H2 SO4 solution. Thereafter, the entire solution was removed from the sample surface using a 100 μ L automatic pipette and inserted into the wells of a microtiter plate (8 mm × 12 mm). To determine the activity of the HRP, the absorbance was measured at a wavelength of 450 nm with a microplate reader “Mithras LB 940” (Berthold Technologies, Germany). The reader operates with a halogen lamp of 75 W. During measurements the lamp energy was set to 30,000 and the counting time was 0.10 s.
Proteins might bind specifically to a surface by the reaction of lysine amino groups with aldehyde groups present on the surface via imine formation followed by reductive amination. It is the aim of this paper to demonstrate that such an immobilization protocol works effectively and leads to active enzyme bound to the surface. To prepare an aldehyde-modified surface on a gold substrate, firstly (3-mercaptopropyl)trimethoxysilane (3MPTMS) was immobilized on the gold. The methoxysilyl groups of the 3MPTMS were then hydrolyzed and condensed with prehydrolyzed 11(triethoxysilyl)undecanal (TESU) in a sol–gel reaction of the silanol groups of both compounds (Scheme 1). PM-IRRAS was used to follow the reactions. The spectrum of the formed layer clearly shows the signals for the aldehyde group at 1730 cm−1 , the CH2 bending vibrations of the long alkyl chain at 1458 cm−1 , and finally a broad band at 950–1250 cm−1 corresponding to the Si O Si group. As proteins have many functional groups resulting in complicated IR spectra, p-aminophenol was selected as a model compound to demonstrate that the desired surface reaction of imine formation between the surface aldehyde and the amino group followed by reductive amination takes place. Coupling of p-aminophenol to the TESU film was detected by the disappearance of the carbonyl band at 1730 cm−1 . This group is clearly visible in the spectrum of the 3MPTMS/TESU layer (Fig. 1b) and almost disappears after 6 h reaction with the p-aminophenol solution followed by reduction with NaCNBH3 (Fig. 1c). Focusing on absorption bands of groups which do not take part in the proposed reaction (δ(CH2 ,rk)ip at 1458 cm−1 , δ(CH3 ,rk)ip at 1167 cm−1 , and ν(Si O Si) at 1126 cm−1 ), it is notable that the intensity of these bands remains nearly the same (Fig. 1b and c). This indicates the stability of the 3MPTMS/TESU base layer under the selected reaction conditions using a shaker. Generally, the PM-IRRAS spectra confirm successful immobilization of the p-aminophenol to the surface as proposed in Scheme 1.
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Fig. 1. (I) PM-IRRAS spectra in the 1800–1000 cm−1 region for (a) pure p-aminophenol (ATR-IR spectrum), (b) freshly assembled TESU on 3MPTMS film, (c) after 6 h chemical attachment of p-aminophenol on TESU film, (d) after 6 h chemical attachment of HRP on TESU film, (e) HRP directly adsorbed on the gold surface. (II) Magnification of the amide I and II bands of the spectra (d) and (e) together with the amide bands of HRP in aqueous solution. Intensity of amide II decreases in the case of (e) in comparison to amide I.
HRP also couples effectively, with clear evidence for chemical attachment between HRP and aldehyde groups of the TESU layer. Comparison of spectra 1b and 1d shows that the intensity of the ν(C O) vibration of the surface aldehyde at 1730 cm−1 almost disappears, confirming the reaction between the enzyme (HRP) and the aldehyde groups. The stability of the 3MPTMS/TESU layer is again confirmed as no intensity loss of the other bands of this layer is observed (Fig. 1d). Furthermore, the amide I band at 1659 cm−1 and amide II band at 1548 cm−1 of HRP are observed on the TESU modified gold surface and on the pristine gold surface (Fig. 1d and e). The intensity of the amide signals in both spectra is very similar and intensity as well as band structure does not change even if the final washing step with shaking is extended from the usual 120–300 min. This shows that the protein content of the surfaces is similar and that the layers are stable under the selected experimental conditions. The shape of the amide I band of HRP in water, adsorbed on the TESU modified gold surface (covalent immobilization) and on the gold surface (physisorption) is similar. However, the amide II bands in Fig. 1 II are in relation to the amide I bands much less intense in spectrum e (physisorption) compared to spectrum d of covalently immobilized HRP and HRP in water. The shape and intensity of amide I and II are sensitive to the superstructure of a protein and the changes indicate destruction of the protein conformation and hence enzyme activity [33]. From the observed difference between the spectra, different activity is expected as is well known from proteins in solution. Unfortunately, quantification and assignment of the α-helix or β-sheet content is not possible with the calibrations known for proteins in solution as the band intensities in IR spectra taken from surface immobilized molecules are also very dependent on molecule and bond orientation. Nevertheless, the spectra show clear differences when the HRP is immobilized via the developed specific protocol or non-specifically directly on the gold surface.
for the samples with HRP immobilized via the 3MPTMS/TESU layer. This shows that the HRP activity is approximately three-fold higher for the specific immobilization. The activity difference is in agreement with the spectroscopic differences obtained by PM-IRRAS and confirm structure preservation when the HRP is bound via the new immobilization protocol.
3.2. Quantitative analysis of the surface immobilized HRP activity
References
The HRP activity has been studied quantitatively after adsorption of HRP on unmodified gold as well as after chemical attachment of HRP on the 3MPTMS/TESU film. This was carried out for the enzymatic oxidation of 3,3 ,5,5 -tetramethylbenzidine leading to a blue color [15]. The average resulting absorbance from three repetitions is 0.067 for the HRP immobilized directly on gold but as high as 0.242
4. Conclusions An alternative method for covalent immobilization of proteins is presented, using horseradish peroxidase as an example. HRP was used as its activity can be readily followed [15,34]. Surface aldehyde is consumed during immobilization and it is expected that the proteins bind via amino groups, for example from lysine. PM-IRRAS is a versatile tool for following the reactions and shows that the shape of the amide bands differs depending on whether the HRP is bound specifically via the new protocol or bound non-specifically directly on the gold surface. These structural differences correlate with activity differences of the HRP. Unfortunately it is not yet possible to quantitatively correlate spectral changes with structural and activity changes of the immobilized protein. Acknowledgements The authors are grateful for financial support for this research by the German Federal Ministry of Education and Research (BMBF, funding reference: 13N9788). Supplementary Material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2013.07.022.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Influence of immobilization protocol on the structure and function of surface bound proteins Alexej Kreider, Stephan Sell, Thomas Kowalik, Andreas Hartwig* , Ingo Grunwald Fraunhofer Institute for Manufacturing Technology and Advanced Materials, Wiener Strasse 12, Bremen 28359, Germany
a r t i c l e
i n f o
Article history: Received 6 March 2013 Received in revised form 8 July 2013 Accepted 8 July 2013 Keywords: Covalent immobilization Horseradish peroxidase PM-IRRAS Bioactivity Biofunctionalization
a b s t r a c t A new coupling strategy for biomacromolecules with (3-mercaptopropyl)trimethoxysilane (3MPTMS) and 11-(triethoxysilyl)undecanal (TESU) on gold surfaces is. This immobilization protocol was utilized for the enzyme horseradish peroxidase (HRP). To study the reactions and resulting structures, PM-IRRAS measurements were performed. PM-IRRAS shows there is structure preservation of the HRP when the new coupling strategy is used in contrast to non-specific adsorption on gold. The biological activity of adsorbed and immobilized HRP was measured by the enzyme catalyzed oxidation of 3,5,3 ,5 -tetramethylbenzidine. Covalent immobilization of HRP on TESU film compared to physisorption of HRP shows higher enzyme activity on gold surfaces, confirming the structural preservation detected by PM-IRRAS. c 2013 Elsevier B.V. All rights reserved.
1. Introduction Technological advancement in areas such as chemical sensors, biosensors, biochips [1,2], and coating materials [3,4] requires a chemical and physical understanding of surface processes. When biomacromolecules such as enzymes are immobilized on support materials, it is essential to conserve the biological activity and accessibility of the catalytically active sites of the enzymes [5–7]. In this context, the formation of an organic layer is the most widely used strategy for the covalent immobilization of biomolecules on surface materials using self-assembled monolayers [8,9]. The formation of organic monolayer films by self-assembly is a common approach for modification of a variety of metals using alkanethiols [10] and alkoxysilanes [11] with different functional groups. The groups are often carboxyl groups with the disadvantage of the need for activation with N-hydroxysuccinimide (NHS) ester and 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC) [12]. The aim of the work presented here is to introduce an alternative immobilization strategy, using the example of horseradish peroxidase (HRP). The thiosilane (3-mercaptopropyl)trimethoxysilane (3MPTMS) is used to functionalize a gold surface via the mercapto group. The hydrolyzable methoxysilyl groups of the surface immobilized 3MPTMS are used to graft the aldehyde terminated organosilane 11(triethoxysilyl)undecanal (TESU) via a sol–gel reaction on the surface
* Corresponding author. Tel.: +49 04212246470; fax: +49 04212246430. E-mail address: [email protected] (A. Hartwig).
c 2013 Elsevier B.V. All rights reserved. 0927-7765/$ - see front matter http://dx.doi.org/10.1016/j.colsurfb.2013.07.022
[13]. The resulting aldehyde modified surfaces were used to immobilize p-aminophenol as a model molecule or HRP as a biomacromolecule having a known secondary structure and an easily measurable bioactivity. The amine groups of lysine residues from the HRP can react with the surfaces’ aldehyde groups forming a Schiff’s base (imine) which is further reduced via reductive amination to a stable secondary amine using sodium cyanoborohydride NaCNBH3 [14,15]. Water, alcohols, and other oxygen nucleophiles typical for biological fluids do not compromise the yields of reductive amination [16,17]. Scheme 1 illustrates the overall strategy for surface immobilization described in this work. In order to analyze the adsorption phenomena and adhesion processes of biomolecules on the surfaces, a variety of measurement techniques can be used including infrared reflection absorption spectroscopy (IRRAS) [18], time of flight secondary ion mass spectrometry (ToF-SIMS) [19], X-ray photoelectron spectroscopy (XPS) [19], and atomic force microscopy (AFM) [20]. Of these measurement techniques, IRRAS is a particularly sensitive and nondestructive technique for acquiring information about, for example, the formation of chemical bonds with the substrate, the conformation, hydrogen-bonded structures, and the orientation of functional groups in the adsorbed substances on the metal surfaces [21]. IRRAS is only partially suitable for samples with an ultrathin film (thickness less than 10 nm) as the measurement time necessary to get an acceptable signal-tonoise ratio becomes several hours [22] and the long measurement time often leads to spectral artifacts due to small changes in the atmospheric composition, especially the water vapor content. For this reason, PM-IRRAS was developed [23]. The polarization modulation (PM) is a very efficient way of discriminating near-surface absorptions from the isotropic strong absorptions occurring in the sample environment [22]. Due to PM, the sample with thin film is also the
Alexej Kreider et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 378–382
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Scheme 1. Strategy for the immobilization of the biomolecule HRP and the model compound p-aminophenol on the gold surface (H&C = hydrolysis and condensation).
reference sample measured at the same time. Therefore, omnipresent contaminations such as hydrocarbons are also detected. They are often overlooked in conventional IRRAS experiments but their presence is, for example, known from XPS experiments. There are several works that use the enzyme HRP to determine the catalytic activity at interfaces [24–27]. In this study we have also chosen HRP as the model enzyme for the readily detectable oxidation of 3,5,3 5 -tetramethylbenzidine diamine to 3,5,3 5 tetramethylbenzidine diimine (Scheme 2). Josephy et al. [28] described the mechanism of oxidation of 3,5,3 ,5 -tetramethylbenzidine using HRP.
2.2. IR spectra ATR-IR spectra were recorded between 4000 and 650 cm−1 on a Bruker Equinox 55 FT-IR-spectrometer (Bruker Optik, Germany) equipped with Golden GateTM ATR unit A531G (Specac) with a resolution of 4 cm−1 and 32 scans. For the PM-IRRAS experiments a polarization modulation accessory (Bruker PMA-50) was used with MCT detector and a photoelastic modulator controller (PEM-90 controller) from Hinds Instruments (HINDS model II/Zn50 with a nominal frequency of 50 kHz, retardation range for half wave 1–10 μ m, and aperture 14 mm) coupled with a Bruker Tensor 27 FT-IR spectrometer. The spectra were an average of 45 min recording time and were taken at a spectral resolution of 4 cm−1 and an incident angle of 83.5◦ . 2.3. Immobilization protocol
2. Experimental 2.1. Materials Au/SiO2 wafers were obtained from Si-Mat Silicon Materials (diameter 100 mm, orientation P/Bor 1 0 0, thickness 525 ± 25 μ m, single polished). (3-Mercaptopropyl)trimethoxysilane (3MPTMS, 95%) and 11-(triethoxysilyl)undecanal (TESU, 90%) were purchased from ABCR (Karlsruhe, Germany). Horseradish peroxidase (HRP, Pcode: 101161191), triethylamine (TEA, 99.5%), methanol (≥99.9%), ethanol (96%), sodium hydrogen carbonate (≥99%, NaHCO3 ), sodium carbonate (≥99%, Na2 CO3 ), and sodium cyanoborohydride (5M, NaBH3 CN) were purchased from Sigma–Aldrich (Steinbach, Germany). One Step Ultra TMB-ELISA was acquired from Pierce Protein Research Products (ThermoScientific, Bonn, Germany). Water was obtained from an UHQ II ELGA water purification system (18 M/cm).
3MPTMS layers were formed at room temperature (RT) on the gold surface by immersion in a solution of 20 mM 3MPTMS in methanol for 2 h (Scheme 1). Then the silanized substrate was rinsed with methanol for 1 h at a shaking frequency of 100 min−1 using an orbital shaker and dried under a nitrogen stream. The samples were then dried in an exsiccator (200 mbar) for 16 h. The hydrolysis and condensation of the 3MPTMS on the substrates were carried out at 80 ◦ C by immersing the 3MPTMS-modified surfaces in 0.1 M HCl for 1 h, 2 h, and 3 h. Subsequently, the prehydrolyzed 3MPTMS layer was cleaned with double distilled water and dried in a nitrogen stream for about 1 min. The silanization process was carried out by incubating the prehydrolyzed 3MPTMS layer in a 96% ethanol solution containing 2% TESU and 1% TEA for 2 h at RT. Then the surfaces were rinsed with ethanol for 10 min at 100 min−1 using an orbital shaker and heated for 2 h at 110 ◦ C. The TEA acts as catalyst for the hydrolysis and condensation
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Alexej Kreider et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 378–382
Scheme 2. Illustration of the applied enzymatic assay. (A) Oxidation of 3,5,3 ,5 -tetramethylbenzidine using HRP. (B) Covalent immobilization of HRP on the TESU-film. (C) Physical adsorption of HRP on the gold surface.
reaction of TESU with the 3MPTMS layer [29,30].
3. Results and discussion 3.1. Protocol for covalent protein immobilization
2.4. Coupling of p-aminophenol and horseradish peroxidase to the aldehyde functionalized gold surfaces The aldehyde-modified substrates for bonding p-aminophenol and the HRP by reductive amination [31,32] were placed in a 0.05 M carbonate buffer solution (pH 9.6) containing 168 mg/20 mL paminophenol or 2 mg/10 mL HRP for 6 h at RT. Then 800 μ L 5 M NaCNBH3 were added to the solutions to reduce the imine product formed on the surfaces. Reaction was for 3 h at RT. Subsequently, the substrates were rinsed once with carbonate buffer (pH 9.6) and once with double distilled water at 100 min−1 for 2 h using an orbital shaker.
2.5. Determination of the activity of immobilized HRP on the surface The activity of immobilized HRP on the TESU film or nonspecifically directly on the gold surface was determined by measuring the color development based on the 1-Step Ultra TMB-ELISA instruction. 3,3 ,5,5 -Tetramethylbenzidine (TMB) was used as the chromogen. To carry out the experiment one drop with a volume of 50 μ L of 1-Step Ultra TMB-ELISA was placed on the sample surface and incubated in the dark for 15 min at room temperature. The solution turned blue. The reaction was stopped by adding 50 μ L of 2 M H2 SO4 solution. Thereafter, the entire solution was removed from the sample surface using a 100 μ L automatic pipette and inserted into the wells of a microtiter plate (8 mm × 12 mm). To determine the activity of the HRP, the absorbance was measured at a wavelength of 450 nm with a microplate reader “Mithras LB 940” (Berthold Technologies, Germany). The reader operates with a halogen lamp of 75 W. During measurements the lamp energy was set to 30,000 and the counting time was 0.10 s.
Proteins might bind specifically to a surface by the reaction of lysine amino groups with aldehyde groups present on the surface via imine formation followed by reductive amination. It is the aim of this paper to demonstrate that such an immobilization protocol works effectively and leads to active enzyme bound to the surface. To prepare an aldehyde-modified surface on a gold substrate, firstly (3-mercaptopropyl)trimethoxysilane (3MPTMS) was immobilized on the gold. The methoxysilyl groups of the 3MPTMS were then hydrolyzed and condensed with prehydrolyzed 11(triethoxysilyl)undecanal (TESU) in a sol–gel reaction of the silanol groups of both compounds (Scheme 1). PM-IRRAS was used to follow the reactions. The spectrum of the formed layer clearly shows the signals for the aldehyde group at 1730 cm−1 , the CH2 bending vibrations of the long alkyl chain at 1458 cm−1 , and finally a broad band at 950–1250 cm−1 corresponding to the Si O Si group. As proteins have many functional groups resulting in complicated IR spectra, p-aminophenol was selected as a model compound to demonstrate that the desired surface reaction of imine formation between the surface aldehyde and the amino group followed by reductive amination takes place. Coupling of p-aminophenol to the TESU film was detected by the disappearance of the carbonyl band at 1730 cm−1 . This group is clearly visible in the spectrum of the 3MPTMS/TESU layer (Fig. 1b) and almost disappears after 6 h reaction with the p-aminophenol solution followed by reduction with NaCNBH3 (Fig. 1c). Focusing on absorption bands of groups which do not take part in the proposed reaction (δ(CH2 ,rk)ip at 1458 cm−1 , δ(CH3 ,rk)ip at 1167 cm−1 , and ν(Si O Si) at 1126 cm−1 ), it is notable that the intensity of these bands remains nearly the same (Fig. 1b and c). This indicates the stability of the 3MPTMS/TESU base layer under the selected reaction conditions using a shaker. Generally, the PM-IRRAS spectra confirm successful immobilization of the p-aminophenol to the surface as proposed in Scheme 1.
Alexej Kreider et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 378–382
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Fig. 1. (I) PM-IRRAS spectra in the 1800–1000 cm−1 region for (a) pure p-aminophenol (ATR-IR spectrum), (b) freshly assembled TESU on 3MPTMS film, (c) after 6 h chemical attachment of p-aminophenol on TESU film, (d) after 6 h chemical attachment of HRP on TESU film, (e) HRP directly adsorbed on the gold surface. (II) Magnification of the amide I and II bands of the spectra (d) and (e) together with the amide bands of HRP in aqueous solution. Intensity of amide II decreases in the case of (e) in comparison to amide I.
HRP also couples effectively, with clear evidence for chemical attachment between HRP and aldehyde groups of the TESU layer. Comparison of spectra 1b and 1d shows that the intensity of the ν(C O) vibration of the surface aldehyde at 1730 cm−1 almost disappears, confirming the reaction between the enzyme (HRP) and the aldehyde groups. The stability of the 3MPTMS/TESU layer is again confirmed as no intensity loss of the other bands of this layer is observed (Fig. 1d). Furthermore, the amide I band at 1659 cm−1 and amide II band at 1548 cm−1 of HRP are observed on the TESU modified gold surface and on the pristine gold surface (Fig. 1d and e). The intensity of the amide signals in both spectra is very similar and intensity as well as band structure does not change even if the final washing step with shaking is extended from the usual 120–300 min. This shows that the protein content of the surfaces is similar and that the layers are stable under the selected experimental conditions. The shape of the amide I band of HRP in water, adsorbed on the TESU modified gold surface (covalent immobilization) and on the gold surface (physisorption) is similar. However, the amide II bands in Fig. 1 II are in relation to the amide I bands much less intense in spectrum e (physisorption) compared to spectrum d of covalently immobilized HRP and HRP in water. The shape and intensity of amide I and II are sensitive to the superstructure of a protein and the changes indicate destruction of the protein conformation and hence enzyme activity [33]. From the observed difference between the spectra, different activity is expected as is well known from proteins in solution. Unfortunately, quantification and assignment of the α-helix or β-sheet content is not possible with the calibrations known for proteins in solution as the band intensities in IR spectra taken from surface immobilized molecules are also very dependent on molecule and bond orientation. Nevertheless, the spectra show clear differences when the HRP is immobilized via the developed specific protocol or non-specifically directly on the gold surface.
for the samples with HRP immobilized via the 3MPTMS/TESU layer. This shows that the HRP activity is approximately three-fold higher for the specific immobilization. The activity difference is in agreement with the spectroscopic differences obtained by PM-IRRAS and confirm structure preservation when the HRP is bound via the new immobilization protocol.
3.2. Quantitative analysis of the surface immobilized HRP activity
References
The HRP activity has been studied quantitatively after adsorption of HRP on unmodified gold as well as after chemical attachment of HRP on the 3MPTMS/TESU film. This was carried out for the enzymatic oxidation of 3,3 ,5,5 -tetramethylbenzidine leading to a blue color [15]. The average resulting absorbance from three repetitions is 0.067 for the HRP immobilized directly on gold but as high as 0.242
4. Conclusions An alternative method for covalent immobilization of proteins is presented, using horseradish peroxidase as an example. HRP was used as its activity can be readily followed [15,34]. Surface aldehyde is consumed during immobilization and it is expected that the proteins bind via amino groups, for example from lysine. PM-IRRAS is a versatile tool for following the reactions and shows that the shape of the amide bands differs depending on whether the HRP is bound specifically via the new protocol or bound non-specifically directly on the gold surface. These structural differences correlate with activity differences of the HRP. Unfortunately it is not yet possible to quantitatively correlate spectral changes with structural and activity changes of the immobilized protein. Acknowledgements The authors are grateful for financial support for this research by the German Federal Ministry of Education and Research (BMBF, funding reference: 13N9788). Supplementary Material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2013.07.022.
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