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A comparative evaluation of molecular recognition by monolayers composed of synthetic receptors or oriented antibodies
Biosensors and Bioelectronics 24 (2008) 1036–1038
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
A comparative evaluation of molecular recognition by monolayers composed of synthetic receptors or oriented antibodies Inger Vikholm-Lundin ∗ , Timo Pulli, Willem M. Albers, Kirsi Tappura VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere, Finland
a r t i c l e
i n f o
Article history: Received 31 March 2008 Received in revised form 4 June 2008 Accepted 24 June 2008 Available online 9 July 2008 Keywords: Immobilisation Antibody Fab fragment Protein repellent polymer Immunoassay Surface plasmon resonance Imprinted self-assembled monolayer
a b s t r a c t Recombinant anti-morphine Fab fragments have been immobilised on gold by covalent attachment through the free thiol groups of the fragment. The antibody fragments were intercalated with a non-ionic hydrophilic polymer in order to suppress non-specific binding of interfering substances. The antibodies are oriented on the surface due to the thiol groups of the antibody and the layer shows a high response to antigen. Non-specific binding of bovine serum albumin is moreover very low because of the repellent polymer. Synthetic receptors composed of an imprinted self-assembled monolayer made from lipoates and the template, morphine, exhibit the same binding response to the antigen, morphine as the site-specific oriented antibody monolayer. A similar binding curve could be obtained as that for binding of morphine to an antibody Fab fragment/polymer layer – indicating that synthetic receptors produced are comparable to those of antibody layers. Concentrations down to 0.1 ng/ml have been measured with surface plasmon resonance. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Antibodies, which can be elicited against essentially any molecule foreign to the host, are widely used as specific capture molecules in immunoassays. Considerable effort has been taken to replace the antibodies with robust and inexpensive synthetic receptors by molecular imprinting polymers (MIPs) (Wulff, 1995; Lahav et al., 1999; Sellergren, 2001). Molecular recognition properties are due to cavities in a highly stable polymer matrix. High sensitivities and selectivities similar to that of natural antibodies have been reported (Yan et al., 2007; Vlatakis et al., 1993). No direct comparative study between layers containing synthetic receptors and antibody molecules has, however, yet been made. In this short communication we will compare the morphine recognition of recombinant antibody Fab fragments/polymer layers with that of imprinted self-assembled monolayers (i-SAMs). Immunoassays are typically prepared by immobilising antibodies through direct physical adsorption on the surface or through covalent coupling onto linking layers via amino and carboxyl groups that are not site-specific. The sensitivity of the assay is highly dependent on the orientation of the immobilised antibodies and on their binding capability (Rao et al., 1998). We have recently shown that human IgG antibody Fab fragments can be directly coupled
∗ Corresponding author. Tel.: +358 40 5389484; fax: +358 20 7223319. E-mail address: inger.vikholm-lundin@vtt.fi (I. Vikholm-Lundin). 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.06.049
onto gold and the space in between the fragments can be filled with non-ionic, hydrophilic disulphide bearing polymers that suppress non-specific binding (Vikholm, 2005; Vikholm-Lundin, 2005). A high degree of the antibody fragments appear to be site-directly immobilised through the free sulphydryl group opposite the antigen binding domain, if proper concentrations of polymer and Fab fragments used for immobilisation onto the gold surface (VikholmLundin, 2005). Coupling of the antibody Fab fragments onto the sensor surface is simple and fast and can be used for attaching any antibody in an oriented manner to the sensor surface. The immobilisation method has also been demonstrated for antibody fragments specific for C reactive protein (Vikholm-Lundin and Albers, 2006). The layer shows a 5-fold higher response to antigen than antibody F(ab)2 -fragments that are randomly adsorbed on the sensor surface. Our approach to produce synthetic receptors has been to use versatile, bifunctional self-assembling ligands, which in the presence of a template are capable of forming i-SAMs (Tappura et al., 2007). The compounds carry a disulphide moiety on one side for anchoring to the gold surface and a variable functional group on the carboxylic acid side for complexation with the template. After selfassembly, the template is washed out of the self-assembled film, and “footprints” may be formed in the layer, which allow rebinding of the template. In a preliminary study morphine was chosen as the model template and various lipoate derivatives were used for i-SAM formation. A large collection of lipoate derivatives was screened by molecular dynamics simulations in various solvents
I. Vikholm-Lundin et al. / Biosensors and Bioelectronics 24 (2008) 1036–1038
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and a set of ligands showing favourable interactions with morphine in aqueous environment was selected for synthesis. The binding of some of these ligands and morphine to gold, as well as the association/dissociation of morphine to the formed layers were studied by surface plasmon resonance, SPR. Imprinting factors as high as 100 and 600 were obtained for one of the ligands – factors 10-fold that normally observed for MIPs (Tappura et al., 2007). 2. Materials and methods 2.1. Production of lipoate, polymer and recombinant anti-morphine antibody Fab fragment The lipoate derivative Lipa-DEA used in this study to prepare iSAMs was made by conjugation of lipoic acid and diethanolamine (DEA) (Tappura et al., 2007). The non-ionic hydrophilic polymer of N-[tris(hydroxymethyl)methyl]acrylamide, pTHMMAA used to suppress non-specific binding to antibody layers were prepared as previously described (Vikholm-Lundin and Albers, 2006). Recombinant anti-morphine M1 antibody Fab fragments has been enriched from phage display library as described previously (Pulli et al., 2005). There were no free cysteines in the Fab fragments to be used for a covalent binding to gold. In order to facilitate the oriented immobilisation of M1 Fab to the sensor surface, unpaired cysteine was cloned to the carboxy terminus of the heavy chain. DNA encoding amino acids DKTHTC was cloned after the 3 end cysteine of the heavy chain constant domain. M1 Fab fragments were expressed in E. coli RV308 strain in 2 l fermentation. Expression was induced by IPTG and cells were grown for 20 h at +30 ◦ C. Cells were centrifuged and Fab fragments were purified from filtered and DNase treated supernatant by Sepharose SP ion-exchange chromatography. The purity and activity of the Fab fragment were checked by SDS-PAGE and ELISA, respectively.
Fig. 2. Binding of 0.1 and 10 ng/ml morphine (lower and upper curve, respectively) to a monolayer composed of antibody Fab fragments and pTHMMAA: (a) denotes injection of morphine and (b) rinsing with buffer.
gold surface typically for 10 min. This was followed by rinsing of the surface with buffer for 5 min. 2.3. Immobilisation of antibody Fab fragments For production of the antibody/polymer layers the same cleaning procedure and assembling of the gold slide as for the i-SAM layer was used. Anti-morphine Fab fragments at a concentration of 50 g/ml of were injected into the Biacore flow-cell for 5–10 min. The surface was hereafter rinsed with the starting buffer, 50 mM Na2 HPO4 /NaH2 PO4 and 150 mM NaCl followed by injection of the polymer, pTHMMAA. The surface was again rinsed for 6 min with buffer and the non-specific binding of BSA and the response to increasing concentrations of morphine in PBS was measured.
2.2. Imprinted self-assembled layer formation 3. Results and discussion The i-SAM formation and the interaction of morphine with the layers were studied by SPR using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). A thin glass slide with an adhesion layer of indium–tin oxide coated with a 50 nm thick film of gold by sputter coating (using an Edwards E306A sputter coater) was cleaned in a hot solution of hydrogen peroxide and ammonium hydroxide in water (1:1:5 v/v/v) and rinsed with high purity water. The slide was mounted in a plastic chip cassette and inserted into the Biacore SPR instrument. The slide was rinsed with 10 mM HEPES, 150 mM NaCl buffer pH 7.5 at a constant flow rate of 20 l/min for one minute. Lipa-DEA was dissolved in ethanol and diluted in HEPES buffer to a ratio of ethanol/HEPES 1:10. Mixed solutions of Lipa-DEA of increasing concentrations (from 0.01 g/l to 1 g/l) and the template (1 mM morphine) were introduced into the microfluidic system of the Biacore instrument, which allowed the compounds to interact with the
3.1. Antibody Fab fragments and polymer monolayers Anti-morphine Fab fragments assemble on the gold surface with a response corresponding to 3060 ± 120 RU. The assembling is very fast and only a minor amount was removed in the dissociation phase. If F(ab)2 fragments are introduced, a considerable amount is removed during rinsing with buffer (Vikholm-Lundin and Albers, 2006). The amount of antibody fragments adsorbed depends on the concentration of the antibody solution (Vikholm-Lundin and Albers, 2006). In order to block the free space remaining on the surface, the polymer, pTHMMAA, was injected over the surface and an additional increase in response corresponding to 1060 ± 70 RU was observed. Part of the antibody fragments might be removed from the surface and replaced by pTHMMAA molecules during the
Fig. 1. Schematic view of morphine binding to (A) an antibody M1 Fab fragment/polymer layer and (B) an imprinted self-assembled monolayer. (C) A schematic drawing of the lipoate derivative, Lipa-DEA.
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I. Vikholm-Lundin et al. / Biosensors and Bioelectronics 24 (2008) 1036–1038
SAM. A schematic view of the layer is given in Fig. 1b. Morphine could be removed from the i-SAM by rinsing the layer with 50 mM NaOH and rebound into the layer. The response of the film to morphine was linear with the logarithm of concentration in the range of 0.1–0.1 g/ml morphine (Fig. 3). A Lipa-DEA non-imprinted SAM showed a response of only 1 ± 10 RU to morphine. The binding of morphine to the i-SAM layer was comparable to that of the antibody Fab fragment/polymer layer (Fig. 3). The non-specific binding of BSA to both layers was very low and the standard curve for binding of morphine was nearly identical. Thus it is evident that synthetic receptors could be produced on the sensor surface according to the i-SAM procedure. These synthetic receptors show a binding to antigen similar to that of very sensitive site-directed monolayers composed of antibody fragments. The i-SAM was not selective to molecules with very similar structure as a high response was also obtained on binding of codeine. But this is neither the case for the antibody fragment that needs a secondary antibody to verify the selectivity (Pulli et al., 2005). 4. Conclusions Fig. 3. Morphine binding to a Lipa-DEA i-SAM () prepared from increasing concentrations of the ligand and antibody M1 Fab fragment/polymer layer () (n = 4).
post-treatment. A schematic view of the layer is given in Fig. 1a. The amount of BSA adsorbed on the binary monolayer composed of antibody Fab fragments intercalated by the polymer was only 15 ± 25 RU, indicating the good repellent properties against nonspecific binding. There was a high binding of various concentrations of morphine to the layer (Figs. 2 and 3). When morphine at a concentration of 0.1 ng/ml interacts with the layer a response of 60 ± 40 RU could be observed, indicating that very low concentrations could easily be measured. At a high concentration of 1 g/ml the response was up to 790 ± 110 RU. It seems that morphine is able to induce higher refractive index changes in the binding layer than what could be deduced by its size. The optical properties of morphine itself or, more probably, the complexes it forms with the surface layer differ significantly from that of a protein. The non-specific binding of BSA corresponded to less than 2%. 3.2. Synthetic receptors – imprinted self-assembled monolayers The lipoate Lipa-DEA forms a monolayer with a response of 1300 ± 750 RU when injected over a clean gold surface. If the template morphine was included in the spreading solution the surface coverage of the i-SAMs corresponded to 2190 ± 170 RU. Morphine corresponding to about 890 RU was thus incorporated in the i-
Imprinted self-assembled monolayers made from lipoates and the template, morphine, show, the same binding response to various concentration of morphine as that of an anti-morphine Fab -fragments/polymer layer site-directly immobilised on gold. Synthetic receptors with binding characteristics similar to those of antibody layers can thus be prepared by i-SAMs. Acknowledgements This work was funded by the EC (project ‘PISARRO’, IST2001-33326), by the Finnish Funding Agency for Technology and Innovation, TEKES (40051/06) and by VTT. References Lahav, M., Katz, E., Doron, A., Patolsky, F., Willner, I., 1999. J. Am. Chem. Soc. 121, 862–863. Pulli, T., Höyhtyä, M., Söderlund, H., Takkinen, K., 2005. Anal. Chem. 77, 2637–2642. Rao, S.V., Anderson, K.W., Bachas, L.G., 1998. Mikrochim. Acta 128, 127–143. Sellergren, B. (Ed.), 2001. Molecularly Imprinted Polymers: Man-made Mimics of Antibodies and their Application in Analytical Chemistry. Elsevier, Amsterdam. Tappura, K., Vikholm-Lundin, I., Albers, W.M., 2007. Biosens. Bioelectron. 22, 912–919. Vikholm, I., 2005. Sens. Actuators B: Chem. 105, 311–316. Vikholm-Lundin, I., 2005. Langmuir 21, 6473–6477. Vikholm-Lundin, I., Albers, W.M., 2006. Biosens. Bioelectron. 21, 1141–1148. Vlatakis, G., Andersson, L.I., Müller, R., Mosbach, K., 1993. Nature 361, 645–647. Wulff, G., 1995. Angew. Chem.: Int. Ed. Engl. 34, 1812–1832. Yan, S., Fang, Y., Gao, Z., 2007. Biosensors and Bioelectronics 22, 1087–1091.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
A comparative evaluation of molecular recognition by monolayers composed of synthetic receptors or oriented antibodies Inger Vikholm-Lundin ∗ , Timo Pulli, Willem M. Albers, Kirsi Tappura VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere, Finland
a r t i c l e
i n f o
Article history: Received 31 March 2008 Received in revised form 4 June 2008 Accepted 24 June 2008 Available online 9 July 2008 Keywords: Immobilisation Antibody Fab fragment Protein repellent polymer Immunoassay Surface plasmon resonance Imprinted self-assembled monolayer
a b s t r a c t Recombinant anti-morphine Fab fragments have been immobilised on gold by covalent attachment through the free thiol groups of the fragment. The antibody fragments were intercalated with a non-ionic hydrophilic polymer in order to suppress non-specific binding of interfering substances. The antibodies are oriented on the surface due to the thiol groups of the antibody and the layer shows a high response to antigen. Non-specific binding of bovine serum albumin is moreover very low because of the repellent polymer. Synthetic receptors composed of an imprinted self-assembled monolayer made from lipoates and the template, morphine, exhibit the same binding response to the antigen, morphine as the site-specific oriented antibody monolayer. A similar binding curve could be obtained as that for binding of morphine to an antibody Fab fragment/polymer layer – indicating that synthetic receptors produced are comparable to those of antibody layers. Concentrations down to 0.1 ng/ml have been measured with surface plasmon resonance. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Antibodies, which can be elicited against essentially any molecule foreign to the host, are widely used as specific capture molecules in immunoassays. Considerable effort has been taken to replace the antibodies with robust and inexpensive synthetic receptors by molecular imprinting polymers (MIPs) (Wulff, 1995; Lahav et al., 1999; Sellergren, 2001). Molecular recognition properties are due to cavities in a highly stable polymer matrix. High sensitivities and selectivities similar to that of natural antibodies have been reported (Yan et al., 2007; Vlatakis et al., 1993). No direct comparative study between layers containing synthetic receptors and antibody molecules has, however, yet been made. In this short communication we will compare the morphine recognition of recombinant antibody Fab fragments/polymer layers with that of imprinted self-assembled monolayers (i-SAMs). Immunoassays are typically prepared by immobilising antibodies through direct physical adsorption on the surface or through covalent coupling onto linking layers via amino and carboxyl groups that are not site-specific. The sensitivity of the assay is highly dependent on the orientation of the immobilised antibodies and on their binding capability (Rao et al., 1998). We have recently shown that human IgG antibody Fab fragments can be directly coupled
∗ Corresponding author. Tel.: +358 40 5389484; fax: +358 20 7223319. E-mail address: inger.vikholm-lundin@vtt.fi (I. Vikholm-Lundin). 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.06.049
onto gold and the space in between the fragments can be filled with non-ionic, hydrophilic disulphide bearing polymers that suppress non-specific binding (Vikholm, 2005; Vikholm-Lundin, 2005). A high degree of the antibody fragments appear to be site-directly immobilised through the free sulphydryl group opposite the antigen binding domain, if proper concentrations of polymer and Fab fragments used for immobilisation onto the gold surface (VikholmLundin, 2005). Coupling of the antibody Fab fragments onto the sensor surface is simple and fast and can be used for attaching any antibody in an oriented manner to the sensor surface. The immobilisation method has also been demonstrated for antibody fragments specific for C reactive protein (Vikholm-Lundin and Albers, 2006). The layer shows a 5-fold higher response to antigen than antibody F(ab)2 -fragments that are randomly adsorbed on the sensor surface. Our approach to produce synthetic receptors has been to use versatile, bifunctional self-assembling ligands, which in the presence of a template are capable of forming i-SAMs (Tappura et al., 2007). The compounds carry a disulphide moiety on one side for anchoring to the gold surface and a variable functional group on the carboxylic acid side for complexation with the template. After selfassembly, the template is washed out of the self-assembled film, and “footprints” may be formed in the layer, which allow rebinding of the template. In a preliminary study morphine was chosen as the model template and various lipoate derivatives were used for i-SAM formation. A large collection of lipoate derivatives was screened by molecular dynamics simulations in various solvents
I. Vikholm-Lundin et al. / Biosensors and Bioelectronics 24 (2008) 1036–1038
1037
and a set of ligands showing favourable interactions with morphine in aqueous environment was selected for synthesis. The binding of some of these ligands and morphine to gold, as well as the association/dissociation of morphine to the formed layers were studied by surface plasmon resonance, SPR. Imprinting factors as high as 100 and 600 were obtained for one of the ligands – factors 10-fold that normally observed for MIPs (Tappura et al., 2007). 2. Materials and methods 2.1. Production of lipoate, polymer and recombinant anti-morphine antibody Fab fragment The lipoate derivative Lipa-DEA used in this study to prepare iSAMs was made by conjugation of lipoic acid and diethanolamine (DEA) (Tappura et al., 2007). The non-ionic hydrophilic polymer of N-[tris(hydroxymethyl)methyl]acrylamide, pTHMMAA used to suppress non-specific binding to antibody layers were prepared as previously described (Vikholm-Lundin and Albers, 2006). Recombinant anti-morphine M1 antibody Fab fragments has been enriched from phage display library as described previously (Pulli et al., 2005). There were no free cysteines in the Fab fragments to be used for a covalent binding to gold. In order to facilitate the oriented immobilisation of M1 Fab to the sensor surface, unpaired cysteine was cloned to the carboxy terminus of the heavy chain. DNA encoding amino acids DKTHTC was cloned after the 3 end cysteine of the heavy chain constant domain. M1 Fab fragments were expressed in E. coli RV308 strain in 2 l fermentation. Expression was induced by IPTG and cells were grown for 20 h at +30 ◦ C. Cells were centrifuged and Fab fragments were purified from filtered and DNase treated supernatant by Sepharose SP ion-exchange chromatography. The purity and activity of the Fab fragment were checked by SDS-PAGE and ELISA, respectively.
Fig. 2. Binding of 0.1 and 10 ng/ml morphine (lower and upper curve, respectively) to a monolayer composed of antibody Fab fragments and pTHMMAA: (a) denotes injection of morphine and (b) rinsing with buffer.
gold surface typically for 10 min. This was followed by rinsing of the surface with buffer for 5 min. 2.3. Immobilisation of antibody Fab fragments For production of the antibody/polymer layers the same cleaning procedure and assembling of the gold slide as for the i-SAM layer was used. Anti-morphine Fab fragments at a concentration of 50 g/ml of were injected into the Biacore flow-cell for 5–10 min. The surface was hereafter rinsed with the starting buffer, 50 mM Na2 HPO4 /NaH2 PO4 and 150 mM NaCl followed by injection of the polymer, pTHMMAA. The surface was again rinsed for 6 min with buffer and the non-specific binding of BSA and the response to increasing concentrations of morphine in PBS was measured.
2.2. Imprinted self-assembled layer formation 3. Results and discussion The i-SAM formation and the interaction of morphine with the layers were studied by SPR using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). A thin glass slide with an adhesion layer of indium–tin oxide coated with a 50 nm thick film of gold by sputter coating (using an Edwards E306A sputter coater) was cleaned in a hot solution of hydrogen peroxide and ammonium hydroxide in water (1:1:5 v/v/v) and rinsed with high purity water. The slide was mounted in a plastic chip cassette and inserted into the Biacore SPR instrument. The slide was rinsed with 10 mM HEPES, 150 mM NaCl buffer pH 7.5 at a constant flow rate of 20 l/min for one minute. Lipa-DEA was dissolved in ethanol and diluted in HEPES buffer to a ratio of ethanol/HEPES 1:10. Mixed solutions of Lipa-DEA of increasing concentrations (from 0.01 g/l to 1 g/l) and the template (1 mM morphine) were introduced into the microfluidic system of the Biacore instrument, which allowed the compounds to interact with the
3.1. Antibody Fab fragments and polymer monolayers Anti-morphine Fab fragments assemble on the gold surface with a response corresponding to 3060 ± 120 RU. The assembling is very fast and only a minor amount was removed in the dissociation phase. If F(ab)2 fragments are introduced, a considerable amount is removed during rinsing with buffer (Vikholm-Lundin and Albers, 2006). The amount of antibody fragments adsorbed depends on the concentration of the antibody solution (Vikholm-Lundin and Albers, 2006). In order to block the free space remaining on the surface, the polymer, pTHMMAA, was injected over the surface and an additional increase in response corresponding to 1060 ± 70 RU was observed. Part of the antibody fragments might be removed from the surface and replaced by pTHMMAA molecules during the
Fig. 1. Schematic view of morphine binding to (A) an antibody M1 Fab fragment/polymer layer and (B) an imprinted self-assembled monolayer. (C) A schematic drawing of the lipoate derivative, Lipa-DEA.
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I. Vikholm-Lundin et al. / Biosensors and Bioelectronics 24 (2008) 1036–1038
SAM. A schematic view of the layer is given in Fig. 1b. Morphine could be removed from the i-SAM by rinsing the layer with 50 mM NaOH and rebound into the layer. The response of the film to morphine was linear with the logarithm of concentration in the range of 0.1–0.1 g/ml morphine (Fig. 3). A Lipa-DEA non-imprinted SAM showed a response of only 1 ± 10 RU to morphine. The binding of morphine to the i-SAM layer was comparable to that of the antibody Fab fragment/polymer layer (Fig. 3). The non-specific binding of BSA to both layers was very low and the standard curve for binding of morphine was nearly identical. Thus it is evident that synthetic receptors could be produced on the sensor surface according to the i-SAM procedure. These synthetic receptors show a binding to antigen similar to that of very sensitive site-directed monolayers composed of antibody fragments. The i-SAM was not selective to molecules with very similar structure as a high response was also obtained on binding of codeine. But this is neither the case for the antibody fragment that needs a secondary antibody to verify the selectivity (Pulli et al., 2005). 4. Conclusions Fig. 3. Morphine binding to a Lipa-DEA i-SAM () prepared from increasing concentrations of the ligand and antibody M1 Fab fragment/polymer layer () (n = 4).
post-treatment. A schematic view of the layer is given in Fig. 1a. The amount of BSA adsorbed on the binary monolayer composed of antibody Fab fragments intercalated by the polymer was only 15 ± 25 RU, indicating the good repellent properties against nonspecific binding. There was a high binding of various concentrations of morphine to the layer (Figs. 2 and 3). When morphine at a concentration of 0.1 ng/ml interacts with the layer a response of 60 ± 40 RU could be observed, indicating that very low concentrations could easily be measured. At a high concentration of 1 g/ml the response was up to 790 ± 110 RU. It seems that morphine is able to induce higher refractive index changes in the binding layer than what could be deduced by its size. The optical properties of morphine itself or, more probably, the complexes it forms with the surface layer differ significantly from that of a protein. The non-specific binding of BSA corresponded to less than 2%. 3.2. Synthetic receptors – imprinted self-assembled monolayers The lipoate Lipa-DEA forms a monolayer with a response of 1300 ± 750 RU when injected over a clean gold surface. If the template morphine was included in the spreading solution the surface coverage of the i-SAMs corresponded to 2190 ± 170 RU. Morphine corresponding to about 890 RU was thus incorporated in the i-
Imprinted self-assembled monolayers made from lipoates and the template, morphine, show, the same binding response to various concentration of morphine as that of an anti-morphine Fab -fragments/polymer layer site-directly immobilised on gold. Synthetic receptors with binding characteristics similar to those of antibody layers can thus be prepared by i-SAMs. Acknowledgements This work was funded by the EC (project ‘PISARRO’, IST2001-33326), by the Finnish Funding Agency for Technology and Innovation, TEKES (40051/06) and by VTT. References Lahav, M., Katz, E., Doron, A., Patolsky, F., Willner, I., 1999. J. Am. Chem. Soc. 121, 862–863. Pulli, T., Höyhtyä, M., Söderlund, H., Takkinen, K., 2005. Anal. Chem. 77, 2637–2642. Rao, S.V., Anderson, K.W., Bachas, L.G., 1998. Mikrochim. Acta 128, 127–143. Sellergren, B. (Ed.), 2001. Molecularly Imprinted Polymers: Man-made Mimics of Antibodies and their Application in Analytical Chemistry. Elsevier, Amsterdam. Tappura, K., Vikholm-Lundin, I., Albers, W.M., 2007. Biosens. Bioelectron. 22, 912–919. Vikholm, I., 2005. Sens. Actuators B: Chem. 105, 311–316. Vikholm-Lundin, I., 2005. Langmuir 21, 6473–6477. Vikholm-Lundin, I., Albers, W.M., 2006. Biosens. Bioelectron. 21, 1141–1148. Vlatakis, G., Andersson, L.I., Müller, R., Mosbach, K., 1993. Nature 361, 645–647. Wulff, G., 1995. Angew. Chem.: Int. Ed. Engl. 34, 1812–1832. Yan, S., Fang, Y., Gao, Z., 2007. Biosensors and Bioelectronics 22, 1087–1091.