- Email: [email protected]
Covalent immobilization of oligonucleotides on electrodes
Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162 www.elsevier.com/locate/colsurfb
Covalent immobilization of oligonucleotides on electrodes Nadia Wrobel a, Werner Deininger b, Peter Hegemann b, Vladimir M. Mirsky a,* a
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany b Institute of Biochemistry, Genetics, and Microbiology, University of Regensburg, D-93040 Regensburg, Germany Received 23 January 2003; accepted 28 May 2003
Abstract 32
P-labeled DNA oligomers were immobilized onto self-assembled monolayers formed by carboxy-modified alkylthiols on gold electrodes. The optimal conditions were evaluated by comparing of surface density of oligonucleotides measured for different immobilization conditions before and after desorption of physically adsorbed nucleotides. The results demonstrate that the physical adsorption depends strongly on immobilization conditions being typically 10 times higher than the chemical immobilization. Under optimal immobilization conditions, the surface density of oligonucleotides was more than 20% of the value calculated for a densely packed monolayer. # 2003 Elsevier B.V. All rights reserved. Keywords: DNA; DNA immobilization; DNA sensor; Biosensor; Hybridization
1. Introduction The design and assembly of sensors for DNA detection and analysis is the basic prerequisite for many applications, including identification of pathogens, monitoring of gene expression, diagnosis of genetic disorders, forensic or pharmaceutic applications, as well as product and food control. Numerous methods and devices used for this purpose [1,2] include for example fluorescence [3], surface plasmon resonance [4], acoustic transduction [5,6], and various electrochemical methods
* Corresponding author. Tel.: /49-941-943-4064; fax: /49941-943-4011. E-mail address: [email protected] (V.M. Mirsky).
[7 /11]. The latter methods have been developed essentially during the last few years [8,11 /15]. For many of these applications, a self-assembled monolayer (SAM) of alkanethiols provides the coupling between the solid electrode and the receptor molecule, i.e. the oligonucleotide. Thiols, as well as other sulfur containing compounds, are able to form SAMs on gold [16 /19], silver [20], platinum [21], palladium [22], iron [23], mercury [24], gallium arsenide [25] and other solid supports, thus providing an anchoring layer for DNA. Once the SAM of an alkanethiol is formed, it is possible to use the tail groups of the thiols, pointing away from the gold film, to immobilize organic substances (the receptor molecules) via chemical reactions [26 /28]. Different physical aspects of the design of DNA biosensors are
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00155-3
158
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
reviewed in [29]. However, the quality of a DNAbiosensor also depends on the efficiency of the chemical immobilization technique. Many factors can have an effect on the immobilization of the receptor molecules: the properties of the solution in which the reaction is performed, e.g. pH, ionic strength, and substances in the solution leading to side reactions of the coupling reagent [30 /32]. The immobilization conditions have to be optimized for each special application in order to increase the yield of chemically attached molecules. In the case of proteins, the empirically determined optimal pH is usually near the isoelectric point or slightly more acidic [28,33]. The chemistry of nucleic acids is less variable than in the case of proteins; therefore the immobilization conditions, once optimized, should be applicable to all DNA molecules. For numerous applications in hybridization sensors, aminomodified DNA oligomers are most often used to obtain the immobilized receptor layer. However, in contrast to proteins, amino-modified DNA oligomers possess only one functional group, making the yield of the immobilization reaction rather small. An essential improvement can be reached by minimization of side reactions [34]. In the present paper we perform a direct optimization of immobilization procedures by means of radiolabeled DNA oligomers.
2. Materials and methods Silicon plates (4 mm /4 mm /0.3 mm) covered by gold on one side, were used as an inorganic substrates for formation of the SAMs of alkanethiols. 16-mercaptohexadecanoic acid purchased from Aldrich was purified by recrystallization. Water was purified in an ion-exchanger purification train (at least 18 MV cm, low organic content; model Millipore plus 185). EDC (1-ethyl-3-(3dimethylaminopropyl)carbodiimide) was from Sigma, S-NHS (N -hydroxysulfosuccinimide) from Aldrich, HEPES (N -(2-hydroxyethyl)piperazine-N ?-(2-ethanesulfonic acid)) from Serva, other chemicals from Merck. All chemicals were used as received. EDC was stored at /18 8C. Lyophilized DNA oligomers modified by an
amino group (combined with a (CH2)6 spacer) were received from MWG Biotech. Measurements were performed on a Beckman LS 6500 multi purpose scintillation counter. The plates, immersed in ethanol (p.a. quality), were treated in an ultrasonic bath twice for 5 min and dried in a stream of nitrogen. They were then dipped for 2 min into hot piranha solution (30% H2O2:98% H2SO4 /1:3, v/v) rinsed with copious amounts of water and blown dry in a stream of nitrogen. Caution: piranha solution reacts violently with most organic materials and must be handled with extreme care . The SAM of the alkanethiol was formed by adsorption from a 100 mmol/l solution of 16-mercaptohexadecanoic acid in chloroform (p.a. quality) for at least 12 h at room temperature (229/2 8C). After coating with alkanethiol, the plates were rinsed with chloroform and dried in a stream of nitrogen. Amino-modified DNA oligomers (5? GCA AAG GGT CGT ACA CAT CAT CAT-NH2 3?) were enzymatically labeled with dideoxyadenosine 5?-[a-32P] triphosphate (Amersham Pharmacia Biotech) using a terminal deoxyribonucleotidyl transferase (TdT) and purified. One millimole DNA oligomers had an activity of 0.315 Ci. The plates were placed in individual plastic tubes. The tubes were filled with the corresponding solutions of different pH and ionic strength (Table 1). Then, radiolabeled DNA oligomers were added to each tube (except the sample 8, Table 1) to give Table 1 Experimental conditions used for immobilization No. Electrolyte
pH Other additives
1 2 3 4 5 6 7
2.2 / 4.0 / 6.9 / 9.4 / 5.8 1 mol/l KCl 5.8 10 mmol/l KCl 7.4 150 mmol/l NaCl 100 mmol/l S-NHS 5.8 200 mmol/l S-NHS
8
0.5 mmol/l phosphate buffer 0.5 mmol/l phosphate buffer 0.5 mmol/l phosphate buffer 0.5 mmol/l phosphate buffer Water Water 10 mmol/l HEPES 3.4 mmol/l EDTA Water, external activationa
For each condition under study two experiments with EDC (400 mmol/l, if not stated otherwise) and one experiment without EDC (control sample) were performed. a 800 mmol/l EDC.
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
a concentration of 4 mmol/l. Five minutes later, EDC was added to several tubes, the remaining tubes serving as a control. Two tubes with EDC addition and one tube as a control were used for each immobilization protocol tested. The plates were incubated in a shaker for 90 min. The eight samples used to test an external activation, were first incubated for 30 min in EDC, then transferred to other tubes filled with water. The radiolabeled DNA oligomers were then added to these samples and incubated on a shaker for 90 min. The gold plates were rinsed with copious amounts of water and the scintillation counts in 20 ml water were determined. The back-ground scintillations were determined in similar measurements without gold plates. The subsequent washing of the DNAcoated gold plates was performed by incubation in 0.2 mol/l NaOH for 0.5 h and in 1 mol/l NaCl for 10 h, on a shaker. After each of these washing procedures, the samples were rinsed with water and scintillation measurements were performed.
3. Results and discussion The radioactivity of identical gold electrodes coated by 32P labelled DNA oligomers according to different immobilization protocols (Table 1) is shown in Fig. 1. A quantitative estimation (below)
Fig. 1. Non-specific adsorption of 32P-labeled amino-modified ssDNA (24-mers) onto gold electrodes covered by a SAM of 16mercaptohexadecanoic acid. The number of each column corresponds to the immobilization conditions specified in the corresponding line of the Table 1. The error bars correspond to the deviation.
159
demonstrates that such high values cannot be explained by formation of only one monolayer of radioactively labelled nucleotides. An increase in pH from acidic to neutral results in about a 2.5fold decrease of the scintillation count values; further pH increase shows no effect (Fig. 1, columns 1 /4). This can be explained by electrostatic interactions. At acidic pH, the carboxygroups of the alkylthiol are not charged and the surface is neutral. The charge of DNA in acidic solution is also less or even zero, therefore an electrostatic repulsion between oligonucleotides and the surface is diminished. At neutral pH, the negative charges of DNA phosphate groups prevail over the positive charge of the single aminogroup. The net charge of the oligonucleotides is negative, the surface is negative too, and therefore electrostatic repulsion can hinder DNA adsorption. At alkaline pH, the oligonucleotides become more negatively charged, and the electrostatic repulsion will increase. An increase of the ionic concentration at neutral pH diminishes electrostatic interactions thus providing higher DNA adsorption (Fig. 1, columns 5/6). The same effect can explain higher DNA binding for sample 7 compared with 8. The back surface of the silicon wafer is oxidized, dissociation of surface silanol groups can lead to a negative surface charge, and therefore one can expect a similar pH dependence for DNA adsorption onto this layer. The DNA desorbed in this procedure can be considered as physically adsorbed. For chemical reasons (formation of amide bonds at room temperature in water is hardly possible in the absence of coupling reagents), the remaining DNA should be also considered as physically adsorbed. An increase of the remaining DNA in the presence of coupling reagent was considered as related to chemically immobilized DNA. Assuming that the amount of strongly adsorbed DNA does not depend on the presence of coupling reagents, one can evaluate the best immobilization procedure by comparing the difference of DNA amounts on the surface in the presence and in the absence of coupling reagents. An essential part of the non-specifically adsorbed DNA oligomers can be removed. Two desorption procedures were used. A 30 min
160
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
incubation in 0.2 mol/l NaOH results in a desorption of about 90 /95% of the adsorbed DNA. Highly alkaline solutions keep DNA denatured without the risk of chemical destruction [35], the gold /thiol bond is also stable under these conditions [36,37]. Therefore this decrease in the concentration of radioactively labelled DNA on the electrode surface cannot be explained by the destruction of chemically immobilized DNA or alkanethiols. Probably one of the reasons for high non-specific adsorption of oligonucleotides is selfhybridization caused by inevitable partial complementarity. A further long-time (10 h) incubation in 0.1 mol/l NaCl leads to removal of only about 25% of the remaining DNA. The DNA amount on the surface of samples prepared in the presence of coupling reagents in different conditions (Table 1) after the two washing procedures are shown as columns in Fig. 2. pH changes after EDC additions were probably one reason that the radioactivity levels for samples 7 and 8 were less than in the control samples (lines).
Fig. 2. Relative amounts of 32P-labeled amino-modified ssDNA (24-mers) on gold electrodes covered by a SAM of 16-mercaptohexadecanoic acid after chemical immobilization in different conditions followed by washing in 0.2 mol/l NaOH for 30 min (black columns) and additionally for 10 h in 0.1 mol/l NaCl (white columns). The average values of the control samples (without EDC) are designated by the upper (washing with NaOH) and lower (washing with NaOH and subsequently with NaCl) lines. The number of each column corresponds to the immobilization conditions specified in the corresponding line of the Table 1. The error bars correspond to the deviation.
The height of the columns in Fig. 2 can be used for comparison of different immobilization procedures. It is remarkable, that the amount of immobilized DNA oligomers is about 10/20 times less than that of the non-specifically adsorbed oligomers. No correlation between immobilized and adsorbed amounts of DNA was observed. Surprisingly, the value of oligonucleotides immobilized in the presence of both EDC and NHS was not higher than in the presence of EDC alone (Fig. 2, column 7, 8). The often advised use of solutions with high ionic strength only provides a small improvement of the DNA immobilization (Fig. 2, columns 5, 6). The highest amount of immobilized DNA was observed for coupling at slightly acidic pH (Fig. 2, columns 1 /4). The mean values of the radioactivity levels after these washing procedures in control experiments (without addition of coupling reagents) are shown by two horizontal lines in Fig. 2. An evaluation of this remaining DNA on electrodes allows us to make quantitative estimations. The surface concentration of adsorbed DNA calculated from its specific activity for column 1 in the Fig. 1 corresponds to about 1400 mmol/m2. Assuming that every DNA molecule occupies 3 nm2, one gets a theoretical limit for the densely packed DNA monolayer of 530 nmol/m2, i.e. almost three times less than the maximum value in Fig. 1. This confirms the suggestion of strong non-specific adsorption of DNA during immobilization. The surface concentration of immobilized DNA oligomers in column 2 of Fig. 2 corresponds to 110 nmol/m2. A comparison with the theoretical limit for the densely packed DNA monolayer demonstrates that we have reached a chemical immobilization up to about 21% of this theoretical value. Therefore, such conditions can be recommended for chemical immobilization of oligonucleotides.
Acknowledgements The authors are grateful to Prof. O.S. Wolfbeis for fruitful discussions. N.W. thanks Sy-Lab GmbH for financial support.
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
References [1] E. Palecek, (Ed.), Talanta, Special issue, 56 (2002) 807 / 976. [2] P.A.E. Piunnoand, U.J. Krull, (Eds.), Anal. Chim. Acta, Special issue, 469 (2002) 1 /154. [3] J.R. Epstein, I. Biran, D.R. Walt, Fluorescence-based nucleic acid detection and microarrays, Anal. Chim. Acta 469 (2002) 3 /36. [4] M. Wu, Z. Lin, V.M. Mirsky, O.S. Wolfbeis, Detection of DNA hybridization with surface plasmon resonance biosensor: comparison of immobilization of oligonucleotides of ssDNA and dsDNA. SPIE Proceedings, 4414 (2001) 23 /26. [5] B.A. Cavic, M. Thompson, Interfacial nucleic acid chemistry studied by acoustic shear wave propagation, Anal. Chim. Acta 469 (2002) 101 /113. [6] I. Willner, F. Patolsky, Y. Weizmann, B. Willner, Amplified detection of single-base mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction, Talanta 56 (2002) 847 /856. [7] J. Wang, Electrochemical nucleic biosensors, Anal. Chim. Acta 469 (2002) 63 /71. [8] E. Palecek, R. Kizek, L. Havran, S. Billova, M. Fojta, Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor, Anal. Chim. Acta 469 (2002) 73 /83. [9] F. Patolsky, E. Katz, A. Bardea, I. Willner, Enzyme-linked amplified electrochemical sensing of oligonucleotide / DNA interactions by means of the precipitation of an insoluble product and using impedance spectroscopy, Langmuir 15 (1999) 3703 /3706. [10] H. Berney, J. West, E. Haefele, J. Alderman, W. Lane, J.K. Collins, A DNA diagnostic biosensor: development, characterisation and performance, Sens. Actuators B 68 (2000) 100 /108. [11] W.G. Kuhr, Electrochemical DNA analysis comes of age, Nature Biotechnol. 18 (2000) 1042 /1043. [12] E. Palecˇek, M. Fojta, Detecting DNA hybridization and damage, Anal. Chem. (2001) 75A /83A. [13] E. Palecˇek, M. Fojta, M. Tomschik, J. Wang, Electrochemical biosensors for DNA hybridization and DNA damage, Biosens. Bioelectron. 13 (1998) 621 /628. [14] M. Mascini, I. Palchetti, G. Marrazza, DNA electrochemical biosensors, Fresen. J. Anal. Chem. 369 (2001) 15 / 22. [15] G. Farace, G. Lillie, T. Hianik, P. Payne, P. Vadgama, Reagentless biosensing using electrochemical impedance spectroscopy, Bioelectrochemistry 55 (2002) 1 /3. [16] C.D. Bain, E.B. Troughton, Y.-T. Tao, Y. Evall, G.M. Whitesides, R.G. Nuzzo, Formation of organic thiols from solution onto gold, J. Am. Chem. Soc. 111 (1989) 321 / 335. [17] R.G. Nuzzo, L.H. Dubios, D.L. Allara, Fundamental studies of microscopic wetting on organic surfaces. 1. Formation and structural characterization of a self-con-
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26] [27] [28]
[29]
[30]
[31] [32]
[33]
161
sistent series of polyfunctional organic monolayers, J. Am. Chem. Soc. 112 (1990) 558 /569. H.O. Finklea, Electrochemistry of organized monolayers of related molecules on electrodes, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 19, Dekker, New York, 1996. V.M. Mirsky, New electroanalytical applications of selfassembled monolayers, Trends Anal. Chem. 21 (2002) 439 /450. M.A. Bryant, J.E. Pemberton, Surface Raman scattering of self assembled monolayers formed from 1-alkanethiols at Ag, J. Am. Chem. Soc. 113 (1991) 3629 /3637. T. Mebrahatu, G.M. Berry, B.G. Bravo, S.L. Michelhaugh, M.P. Soriaga, Substate-mediated adsorbate /adsorbate interactions: effect of submonolayer coverage and coadsorbed iodine on the reversible redox of 2,5-dihydroxythiophenol chemisorbed at Au and Pt, Langmuir 4 (1988) 1147 /1151. M. Riepl, V.M. Mirsky, M. Ostblo¨m, B. Liedberg, Selfassembled monolayers of alkylthiols on palladium electrodes. in: Electrochemistry in Molecular and Microscopic Dimensions. 53rd Annual Meeting in Internal Society of Electrochemistry, Dechema, Du¨sseldorf, 2002. L. Cheng, A.B. Bocarsly, S.L. Bernasek, T.A. Ramanarayanan, Interactions of alkanethiols with single crystal iron: the low temperature decomposition of ethanethiol on the Fe (1 0 0) surface, Langmuir 10 (1994) 4542 /4550. M.A. Rampi, O.J. Schueller, G.M. Whitesides, Alkanethiol self-assembled monolayers as the dielectric of capacitors with nanoscale thickness, Appl. Phys. Lett. 72 (1998) 1781 /1783. K. Adlkofer, M. Tanaka, Stable surface coating of gallium arsenide with octadecylthiol monolayers, Langmuir 17 (2001) 4267 /4273. Z. Junhui, C. Hong, Y. Ruifu, DNA based biosensors, Biotechnol. Adv. 1 (1997) 43 /58. G.T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1995. V.M. Mirsky, M. Riepl, O.S. Wolfbeis, Capacitive monitoring of protein immobilization and antigen-antibody reactions on monomolecular alkylthiol films on gold electrodes, Biosens. Bioelectron. 12 (1997) 977 /989. J. Watterson, P.A.E. Piunno, U.J. Krull, Pryctical aspects of interfacial nucleic acid oligomer hybridization for biosensor design, Anal. Chim. Acta 469 (2002) 115 /127. K.L. Carraway, R.B. Triplett, Reaction of carbodiimides with protein sulfhydryl groups, Biochim. Biophys. Acta 200 (1970) 564 /566. F. Kurzer, K. Douraghi-Zadeh, Advances in the chemistry of carbodiimides, Chem. Rev. 67/2 (1967) 107 /153. K.L. Carraway, D.E. Koshland, Jr., Reaction of tyrosine residues in proteins with carbodiimide reagents, Biochim. Biophys. Acta 160 (1968) 272 /274. R.R. Seigel, P. Harder, R. Dahint, M. Grunze, F. Josse, M. Mrksich, G.M. Whitesides, On-line detection of non-
162
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
specific protein adsorption at artificial surfaces, Anal. Chem. 69 (2000) 3321 /3328. [34] N. Wrobel, M. Schinkinger, V.M. Mirsky, A novel ultraviolet assay for testing side reactions of carbodiimides, Anal. Biochem. 305 (2002) 135 /138. [35] K. Kleparnik, Z. Mala, P. Bocek, Fast separation of DNA sequencing fragments in highly alkaline solutions of linear polyacrylamide using electrophoresis in bare silica capillaries, Electrophoresis 22 (2001) 783 /788.
[36] S.-I. Imabayashi, D. Hobara, T. Kakiuchi, Selective replacement of adsorbed alkanethiols on phase-separated binary self-assembled monolayers by electrochemical partial desorption, Langmuir 13 (1997) 4502 /4504. [37] T. Kawaguchi, H. Yasuda, K. Shimazu, Electrochemical quartz crystal microbalance investigation of the reductive desorption of self-assembled monolayers of alkanethiols and mercaptoalkanoic acids on Au, Langmuir 16 (2000) 9830 /9840.
Covalent immobilization of oligonucleotides on electrodes Nadia Wrobel a, Werner Deininger b, Peter Hegemann b, Vladimir M. Mirsky a,* a
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany b Institute of Biochemistry, Genetics, and Microbiology, University of Regensburg, D-93040 Regensburg, Germany Received 23 January 2003; accepted 28 May 2003
Abstract 32
P-labeled DNA oligomers were immobilized onto self-assembled monolayers formed by carboxy-modified alkylthiols on gold electrodes. The optimal conditions were evaluated by comparing of surface density of oligonucleotides measured for different immobilization conditions before and after desorption of physically adsorbed nucleotides. The results demonstrate that the physical adsorption depends strongly on immobilization conditions being typically 10 times higher than the chemical immobilization. Under optimal immobilization conditions, the surface density of oligonucleotides was more than 20% of the value calculated for a densely packed monolayer. # 2003 Elsevier B.V. All rights reserved. Keywords: DNA; DNA immobilization; DNA sensor; Biosensor; Hybridization
1. Introduction The design and assembly of sensors for DNA detection and analysis is the basic prerequisite for many applications, including identification of pathogens, monitoring of gene expression, diagnosis of genetic disorders, forensic or pharmaceutic applications, as well as product and food control. Numerous methods and devices used for this purpose [1,2] include for example fluorescence [3], surface plasmon resonance [4], acoustic transduction [5,6], and various electrochemical methods
* Corresponding author. Tel.: /49-941-943-4064; fax: /49941-943-4011. E-mail address: [email protected] (V.M. Mirsky).
[7 /11]. The latter methods have been developed essentially during the last few years [8,11 /15]. For many of these applications, a self-assembled monolayer (SAM) of alkanethiols provides the coupling between the solid electrode and the receptor molecule, i.e. the oligonucleotide. Thiols, as well as other sulfur containing compounds, are able to form SAMs on gold [16 /19], silver [20], platinum [21], palladium [22], iron [23], mercury [24], gallium arsenide [25] and other solid supports, thus providing an anchoring layer for DNA. Once the SAM of an alkanethiol is formed, it is possible to use the tail groups of the thiols, pointing away from the gold film, to immobilize organic substances (the receptor molecules) via chemical reactions [26 /28]. Different physical aspects of the design of DNA biosensors are
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00155-3
158
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
reviewed in [29]. However, the quality of a DNAbiosensor also depends on the efficiency of the chemical immobilization technique. Many factors can have an effect on the immobilization of the receptor molecules: the properties of the solution in which the reaction is performed, e.g. pH, ionic strength, and substances in the solution leading to side reactions of the coupling reagent [30 /32]. The immobilization conditions have to be optimized for each special application in order to increase the yield of chemically attached molecules. In the case of proteins, the empirically determined optimal pH is usually near the isoelectric point or slightly more acidic [28,33]. The chemistry of nucleic acids is less variable than in the case of proteins; therefore the immobilization conditions, once optimized, should be applicable to all DNA molecules. For numerous applications in hybridization sensors, aminomodified DNA oligomers are most often used to obtain the immobilized receptor layer. However, in contrast to proteins, amino-modified DNA oligomers possess only one functional group, making the yield of the immobilization reaction rather small. An essential improvement can be reached by minimization of side reactions [34]. In the present paper we perform a direct optimization of immobilization procedures by means of radiolabeled DNA oligomers.
2. Materials and methods Silicon plates (4 mm /4 mm /0.3 mm) covered by gold on one side, were used as an inorganic substrates for formation of the SAMs of alkanethiols. 16-mercaptohexadecanoic acid purchased from Aldrich was purified by recrystallization. Water was purified in an ion-exchanger purification train (at least 18 MV cm, low organic content; model Millipore plus 185). EDC (1-ethyl-3-(3dimethylaminopropyl)carbodiimide) was from Sigma, S-NHS (N -hydroxysulfosuccinimide) from Aldrich, HEPES (N -(2-hydroxyethyl)piperazine-N ?-(2-ethanesulfonic acid)) from Serva, other chemicals from Merck. All chemicals were used as received. EDC was stored at /18 8C. Lyophilized DNA oligomers modified by an
amino group (combined with a (CH2)6 spacer) were received from MWG Biotech. Measurements were performed on a Beckman LS 6500 multi purpose scintillation counter. The plates, immersed in ethanol (p.a. quality), were treated in an ultrasonic bath twice for 5 min and dried in a stream of nitrogen. They were then dipped for 2 min into hot piranha solution (30% H2O2:98% H2SO4 /1:3, v/v) rinsed with copious amounts of water and blown dry in a stream of nitrogen. Caution: piranha solution reacts violently with most organic materials and must be handled with extreme care . The SAM of the alkanethiol was formed by adsorption from a 100 mmol/l solution of 16-mercaptohexadecanoic acid in chloroform (p.a. quality) for at least 12 h at room temperature (229/2 8C). After coating with alkanethiol, the plates were rinsed with chloroform and dried in a stream of nitrogen. Amino-modified DNA oligomers (5? GCA AAG GGT CGT ACA CAT CAT CAT-NH2 3?) were enzymatically labeled with dideoxyadenosine 5?-[a-32P] triphosphate (Amersham Pharmacia Biotech) using a terminal deoxyribonucleotidyl transferase (TdT) and purified. One millimole DNA oligomers had an activity of 0.315 Ci. The plates were placed in individual plastic tubes. The tubes were filled with the corresponding solutions of different pH and ionic strength (Table 1). Then, radiolabeled DNA oligomers were added to each tube (except the sample 8, Table 1) to give Table 1 Experimental conditions used for immobilization No. Electrolyte
pH Other additives
1 2 3 4 5 6 7
2.2 / 4.0 / 6.9 / 9.4 / 5.8 1 mol/l KCl 5.8 10 mmol/l KCl 7.4 150 mmol/l NaCl 100 mmol/l S-NHS 5.8 200 mmol/l S-NHS
8
0.5 mmol/l phosphate buffer 0.5 mmol/l phosphate buffer 0.5 mmol/l phosphate buffer 0.5 mmol/l phosphate buffer Water Water 10 mmol/l HEPES 3.4 mmol/l EDTA Water, external activationa
For each condition under study two experiments with EDC (400 mmol/l, if not stated otherwise) and one experiment without EDC (control sample) were performed. a 800 mmol/l EDC.
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
a concentration of 4 mmol/l. Five minutes later, EDC was added to several tubes, the remaining tubes serving as a control. Two tubes with EDC addition and one tube as a control were used for each immobilization protocol tested. The plates were incubated in a shaker for 90 min. The eight samples used to test an external activation, were first incubated for 30 min in EDC, then transferred to other tubes filled with water. The radiolabeled DNA oligomers were then added to these samples and incubated on a shaker for 90 min. The gold plates were rinsed with copious amounts of water and the scintillation counts in 20 ml water were determined. The back-ground scintillations were determined in similar measurements without gold plates. The subsequent washing of the DNAcoated gold plates was performed by incubation in 0.2 mol/l NaOH for 0.5 h and in 1 mol/l NaCl for 10 h, on a shaker. After each of these washing procedures, the samples were rinsed with water and scintillation measurements were performed.
3. Results and discussion The radioactivity of identical gold electrodes coated by 32P labelled DNA oligomers according to different immobilization protocols (Table 1) is shown in Fig. 1. A quantitative estimation (below)
Fig. 1. Non-specific adsorption of 32P-labeled amino-modified ssDNA (24-mers) onto gold electrodes covered by a SAM of 16mercaptohexadecanoic acid. The number of each column corresponds to the immobilization conditions specified in the corresponding line of the Table 1. The error bars correspond to the deviation.
159
demonstrates that such high values cannot be explained by formation of only one monolayer of radioactively labelled nucleotides. An increase in pH from acidic to neutral results in about a 2.5fold decrease of the scintillation count values; further pH increase shows no effect (Fig. 1, columns 1 /4). This can be explained by electrostatic interactions. At acidic pH, the carboxygroups of the alkylthiol are not charged and the surface is neutral. The charge of DNA in acidic solution is also less or even zero, therefore an electrostatic repulsion between oligonucleotides and the surface is diminished. At neutral pH, the negative charges of DNA phosphate groups prevail over the positive charge of the single aminogroup. The net charge of the oligonucleotides is negative, the surface is negative too, and therefore electrostatic repulsion can hinder DNA adsorption. At alkaline pH, the oligonucleotides become more negatively charged, and the electrostatic repulsion will increase. An increase of the ionic concentration at neutral pH diminishes electrostatic interactions thus providing higher DNA adsorption (Fig. 1, columns 5/6). The same effect can explain higher DNA binding for sample 7 compared with 8. The back surface of the silicon wafer is oxidized, dissociation of surface silanol groups can lead to a negative surface charge, and therefore one can expect a similar pH dependence for DNA adsorption onto this layer. The DNA desorbed in this procedure can be considered as physically adsorbed. For chemical reasons (formation of amide bonds at room temperature in water is hardly possible in the absence of coupling reagents), the remaining DNA should be also considered as physically adsorbed. An increase of the remaining DNA in the presence of coupling reagent was considered as related to chemically immobilized DNA. Assuming that the amount of strongly adsorbed DNA does not depend on the presence of coupling reagents, one can evaluate the best immobilization procedure by comparing the difference of DNA amounts on the surface in the presence and in the absence of coupling reagents. An essential part of the non-specifically adsorbed DNA oligomers can be removed. Two desorption procedures were used. A 30 min
160
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
incubation in 0.2 mol/l NaOH results in a desorption of about 90 /95% of the adsorbed DNA. Highly alkaline solutions keep DNA denatured without the risk of chemical destruction [35], the gold /thiol bond is also stable under these conditions [36,37]. Therefore this decrease in the concentration of radioactively labelled DNA on the electrode surface cannot be explained by the destruction of chemically immobilized DNA or alkanethiols. Probably one of the reasons for high non-specific adsorption of oligonucleotides is selfhybridization caused by inevitable partial complementarity. A further long-time (10 h) incubation in 0.1 mol/l NaCl leads to removal of only about 25% of the remaining DNA. The DNA amount on the surface of samples prepared in the presence of coupling reagents in different conditions (Table 1) after the two washing procedures are shown as columns in Fig. 2. pH changes after EDC additions were probably one reason that the radioactivity levels for samples 7 and 8 were less than in the control samples (lines).
Fig. 2. Relative amounts of 32P-labeled amino-modified ssDNA (24-mers) on gold electrodes covered by a SAM of 16-mercaptohexadecanoic acid after chemical immobilization in different conditions followed by washing in 0.2 mol/l NaOH for 30 min (black columns) and additionally for 10 h in 0.1 mol/l NaCl (white columns). The average values of the control samples (without EDC) are designated by the upper (washing with NaOH) and lower (washing with NaOH and subsequently with NaCl) lines. The number of each column corresponds to the immobilization conditions specified in the corresponding line of the Table 1. The error bars correspond to the deviation.
The height of the columns in Fig. 2 can be used for comparison of different immobilization procedures. It is remarkable, that the amount of immobilized DNA oligomers is about 10/20 times less than that of the non-specifically adsorbed oligomers. No correlation between immobilized and adsorbed amounts of DNA was observed. Surprisingly, the value of oligonucleotides immobilized in the presence of both EDC and NHS was not higher than in the presence of EDC alone (Fig. 2, column 7, 8). The often advised use of solutions with high ionic strength only provides a small improvement of the DNA immobilization (Fig. 2, columns 5, 6). The highest amount of immobilized DNA was observed for coupling at slightly acidic pH (Fig. 2, columns 1 /4). The mean values of the radioactivity levels after these washing procedures in control experiments (without addition of coupling reagents) are shown by two horizontal lines in Fig. 2. An evaluation of this remaining DNA on electrodes allows us to make quantitative estimations. The surface concentration of adsorbed DNA calculated from its specific activity for column 1 in the Fig. 1 corresponds to about 1400 mmol/m2. Assuming that every DNA molecule occupies 3 nm2, one gets a theoretical limit for the densely packed DNA monolayer of 530 nmol/m2, i.e. almost three times less than the maximum value in Fig. 1. This confirms the suggestion of strong non-specific adsorption of DNA during immobilization. The surface concentration of immobilized DNA oligomers in column 2 of Fig. 2 corresponds to 110 nmol/m2. A comparison with the theoretical limit for the densely packed DNA monolayer demonstrates that we have reached a chemical immobilization up to about 21% of this theoretical value. Therefore, such conditions can be recommended for chemical immobilization of oligonucleotides.
Acknowledgements The authors are grateful to Prof. O.S. Wolfbeis for fruitful discussions. N.W. thanks Sy-Lab GmbH for financial support.
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
References [1] E. Palecek, (Ed.), Talanta, Special issue, 56 (2002) 807 / 976. [2] P.A.E. Piunnoand, U.J. Krull, (Eds.), Anal. Chim. Acta, Special issue, 469 (2002) 1 /154. [3] J.R. Epstein, I. Biran, D.R. Walt, Fluorescence-based nucleic acid detection and microarrays, Anal. Chim. Acta 469 (2002) 3 /36. [4] M. Wu, Z. Lin, V.M. Mirsky, O.S. Wolfbeis, Detection of DNA hybridization with surface plasmon resonance biosensor: comparison of immobilization of oligonucleotides of ssDNA and dsDNA. SPIE Proceedings, 4414 (2001) 23 /26. [5] B.A. Cavic, M. Thompson, Interfacial nucleic acid chemistry studied by acoustic shear wave propagation, Anal. Chim. Acta 469 (2002) 101 /113. [6] I. Willner, F. Patolsky, Y. Weizmann, B. Willner, Amplified detection of single-base mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction, Talanta 56 (2002) 847 /856. [7] J. Wang, Electrochemical nucleic biosensors, Anal. Chim. Acta 469 (2002) 63 /71. [8] E. Palecek, R. Kizek, L. Havran, S. Billova, M. Fojta, Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor, Anal. Chim. Acta 469 (2002) 73 /83. [9] F. Patolsky, E. Katz, A. Bardea, I. Willner, Enzyme-linked amplified electrochemical sensing of oligonucleotide / DNA interactions by means of the precipitation of an insoluble product and using impedance spectroscopy, Langmuir 15 (1999) 3703 /3706. [10] H. Berney, J. West, E. Haefele, J. Alderman, W. Lane, J.K. Collins, A DNA diagnostic biosensor: development, characterisation and performance, Sens. Actuators B 68 (2000) 100 /108. [11] W.G. Kuhr, Electrochemical DNA analysis comes of age, Nature Biotechnol. 18 (2000) 1042 /1043. [12] E. Palecˇek, M. Fojta, Detecting DNA hybridization and damage, Anal. Chem. (2001) 75A /83A. [13] E. Palecˇek, M. Fojta, M. Tomschik, J. Wang, Electrochemical biosensors for DNA hybridization and DNA damage, Biosens. Bioelectron. 13 (1998) 621 /628. [14] M. Mascini, I. Palchetti, G. Marrazza, DNA electrochemical biosensors, Fresen. J. Anal. Chem. 369 (2001) 15 / 22. [15] G. Farace, G. Lillie, T. Hianik, P. Payne, P. Vadgama, Reagentless biosensing using electrochemical impedance spectroscopy, Bioelectrochemistry 55 (2002) 1 /3. [16] C.D. Bain, E.B. Troughton, Y.-T. Tao, Y. Evall, G.M. Whitesides, R.G. Nuzzo, Formation of organic thiols from solution onto gold, J. Am. Chem. Soc. 111 (1989) 321 / 335. [17] R.G. Nuzzo, L.H. Dubios, D.L. Allara, Fundamental studies of microscopic wetting on organic surfaces. 1. Formation and structural characterization of a self-con-
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26] [27] [28]
[29]
[30]
[31] [32]
[33]
161
sistent series of polyfunctional organic monolayers, J. Am. Chem. Soc. 112 (1990) 558 /569. H.O. Finklea, Electrochemistry of organized monolayers of related molecules on electrodes, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 19, Dekker, New York, 1996. V.M. Mirsky, New electroanalytical applications of selfassembled monolayers, Trends Anal. Chem. 21 (2002) 439 /450. M.A. Bryant, J.E. Pemberton, Surface Raman scattering of self assembled monolayers formed from 1-alkanethiols at Ag, J. Am. Chem. Soc. 113 (1991) 3629 /3637. T. Mebrahatu, G.M. Berry, B.G. Bravo, S.L. Michelhaugh, M.P. Soriaga, Substate-mediated adsorbate /adsorbate interactions: effect of submonolayer coverage and coadsorbed iodine on the reversible redox of 2,5-dihydroxythiophenol chemisorbed at Au and Pt, Langmuir 4 (1988) 1147 /1151. M. Riepl, V.M. Mirsky, M. Ostblo¨m, B. Liedberg, Selfassembled monolayers of alkylthiols on palladium electrodes. in: Electrochemistry in Molecular and Microscopic Dimensions. 53rd Annual Meeting in Internal Society of Electrochemistry, Dechema, Du¨sseldorf, 2002. L. Cheng, A.B. Bocarsly, S.L. Bernasek, T.A. Ramanarayanan, Interactions of alkanethiols with single crystal iron: the low temperature decomposition of ethanethiol on the Fe (1 0 0) surface, Langmuir 10 (1994) 4542 /4550. M.A. Rampi, O.J. Schueller, G.M. Whitesides, Alkanethiol self-assembled monolayers as the dielectric of capacitors with nanoscale thickness, Appl. Phys. Lett. 72 (1998) 1781 /1783. K. Adlkofer, M. Tanaka, Stable surface coating of gallium arsenide with octadecylthiol monolayers, Langmuir 17 (2001) 4267 /4273. Z. Junhui, C. Hong, Y. Ruifu, DNA based biosensors, Biotechnol. Adv. 1 (1997) 43 /58. G.T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1995. V.M. Mirsky, M. Riepl, O.S. Wolfbeis, Capacitive monitoring of protein immobilization and antigen-antibody reactions on monomolecular alkylthiol films on gold electrodes, Biosens. Bioelectron. 12 (1997) 977 /989. J. Watterson, P.A.E. Piunno, U.J. Krull, Pryctical aspects of interfacial nucleic acid oligomer hybridization for biosensor design, Anal. Chim. Acta 469 (2002) 115 /127. K.L. Carraway, R.B. Triplett, Reaction of carbodiimides with protein sulfhydryl groups, Biochim. Biophys. Acta 200 (1970) 564 /566. F. Kurzer, K. Douraghi-Zadeh, Advances in the chemistry of carbodiimides, Chem. Rev. 67/2 (1967) 107 /153. K.L. Carraway, D.E. Koshland, Jr., Reaction of tyrosine residues in proteins with carbodiimide reagents, Biochim. Biophys. Acta 160 (1968) 272 /274. R.R. Seigel, P. Harder, R. Dahint, M. Grunze, F. Josse, M. Mrksich, G.M. Whitesides, On-line detection of non-
162
N. Wrobel et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 157 /162
specific protein adsorption at artificial surfaces, Anal. Chem. 69 (2000) 3321 /3328. [34] N. Wrobel, M. Schinkinger, V.M. Mirsky, A novel ultraviolet assay for testing side reactions of carbodiimides, Anal. Biochem. 305 (2002) 135 /138. [35] K. Kleparnik, Z. Mala, P. Bocek, Fast separation of DNA sequencing fragments in highly alkaline solutions of linear polyacrylamide using electrophoresis in bare silica capillaries, Electrophoresis 22 (2001) 783 /788.
[36] S.-I. Imabayashi, D. Hobara, T. Kakiuchi, Selective replacement of adsorbed alkanethiols on phase-separated binary self-assembled monolayers by electrochemical partial desorption, Langmuir 13 (1997) 4502 /4504. [37] T. Kawaguchi, H. Yasuda, K. Shimazu, Electrochemical quartz crystal microbalance investigation of the reductive desorption of self-assembled monolayers of alkanethiols and mercaptoalkanoic acids on Au, Langmuir 16 (2000) 9830 /9840.