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An immunosensor for haemoglobin based on impedimetric properties of a new mixed self-assembled monolayer
Materials Science and Engineering C 26 (2006) 322 – 327 www.elsevier.com/locate/msec
An immunosensor for haemoglobin based on impedimetric properties of a new mixed self-assembled monolayer S. Hleli a,b, C. Martelet a,*, A. Abdelghani b, F. Bessueille c, A. Errachid c, J. Samitier c, H.C.W. Hays d, P.A. Millner d, N. Burais a, N. Jaffrezic-Renault a b
a Centre de Ge´nie Electrique de Lyon, CEGELY, ECL, 69134 Ecully cedex, France Unite´ de recherche de physique des semiconducteurs et capteurs, IPEST, La Marsa, Tunisia c Laboratory of NanoBioEngineering, Barcelona Science Park, Barcelona 08028, Spain d School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
Available online 7 December 2005
Abstract A novel impedimetric immunosensor for the detection of haemoglobin has been developed by mixed self-assembled monolayers on Au. First, a mixed self-assembled monolayer (SAMs) consisting of 1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotinyl-PE) and 16mercaptohexadecanoic acid (MHDA) on gold electrodes was studied. The conformational properties of the SAMs were characterised by cyclic voltammetry and impedance spectroscopy. After blocking non-specific binding sites in the mixed monolayer using non-specific IgG, neutravidin was used to bind to biotinyl sites present in the mixed monolayer. Finally, biotinylated anti-haemoglobin IgG was immobilised to the tethered neutravidin. The membrane resistance R m, obtained from the assembly, decreased gradually after the addition of non-specific IgG, neutravidin and anti-haemoglobin to the monolayer. This decrease could be attributed to a rearrangement in the structure of the SAMs. The detection of antibody – antigen reaction demonstrates that the potentiometric immunosensor exhibited high sensitivity and a detection limit of 20 ng/ml (approximately 0.3 nM). D 2005 Elsevier B.V. All rights reserved. Keywords: Human haemoglobin; Biotinyl-PE; Neutravidin; Mixed self-assembled monolayers; Impedance spectroscopy; AFM; Biosensors
1. Introduction The development of numerous immunochemical methods for the measurement of antibody –antigen reactions continues to be the subject of considerable research and development effort [1 – 3]. In the last two decades, many reports have been published on the use of immunosensors for a wide range of applications in food, environment, pharmaceutical chemistry and clinical diagnostics. Immunosensors are based on the use of an antibody that reacts specifically with the substance (antigen) to be tested. Therefore, one of the most important points in the design of an immunosensor is the choice of immobilization method. Recently, self-assembled monolayers (SAMs) generated by the adsorption of alkanethiols on gold have been the subject of extensive research due to their simple preparation and special characteristics. One of the best understood functionalised self* Corresponding author. Tel.: +33 472186248; fax: +33 478433717. E-mail address: [email protected] (C. Martelet). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.10.054
assembled monolayer systems is that based on biotin/streptavidin to attach a sensor biomolecule, which is usually an enzyme or an antibody [4 – 7]. Streptavidin has an extremely high binding affinity for biotin (Ka¨1015M) [8,9] and the streptavidin molecule is a homotetramer with each subunit having a single biotin binding site. Therefore, it can be used as a linker between two biotin tagged components, such as the surface of an electrode and a biotin tagged protein. Among the various transduction techniques, electrochemical impedance spectroscopy (EIS) has previously been investigated to study the selective binding of streptavidin to avidinterminated alkanethiols and disulfide or sulfide containing molecules self-assembled on gold [10 – 13]. EIS is a sensitive technique, which monitors the electrical response of the system studied after application of a periodic small amplitude AC signal. Analysis of the system response provides information concerning the interface and reactions occurring at it [14,15]. EIS has been widely used by several workers to characterise SAM quality [16].
S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
323
In this work, IgG directed against human haemoglobin was immobilised on a mixed self-assembled layer with neutravidin and faradaic impedance spectroscopy was applied as the electrochemical sensing method. A novel mixed self-assembled monolayer consisting of biotinyl-PE and MHDA was examined using electrochemical impedance spectroscopy and cyclic voltammetry. The neutravidin/biotin interaction and conformational effects induced within the SAM were detected using the impedance spectroscopy.
2.4. Immunoassay of haemoglobin
2. Experimental
2.5. Impedance spectroscopy
2.1. Material
The measurement set-up for impedance consisted of a classical three-electrode system, where the modified gold electrode (0.21 cm2) was used as working electrode, a platinum strip (0.54 cm2) as a counter electrode and a saturated calomel electrode (SCE) as reference electrode. The impedance analysis was performed with the Voltalab 80 impedance analyser in the frequency range 0.5 Hz – 100 kHz, using a modulation voltage of 10 mV. During measurements the potential was kept at zero volts. The impedance measurements and cyclic voltametry measurements were performed in the presence of a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] (1 : 1) mixture as redox probe in PBS. The Zview modelling programme (Scriber and associates, Charlottesville, VA) was used. All electrochemical measurements were carried out at room temperature and in a faraday cage.
Immunoglobulin G (IgG) against human haemoglobin was from bethyl and was biotinylated using N-(+)-biotinyl-6aminocaproic acid N-succinimidyl ester according to manufacturers instructions. Human haemoglobin and non-immune goat IgG were purchased from Sigma-Aldrich whilst neutravidin was obtained from Pierce. The affinity phospholipid dipalmitoyl-snglycero-3-phospho-ethanolamine-N-(biotinyl) (biotinyl-PE) and 16-mercaptohexadecanoic acid, for the formation of mixed monolayers were purchased from Avanti polar lipids and SigmaAldrich, respectively. The buffer solution used for all experiments was phosphate buffered saline (PBS) containing 140 mM NaCl, 2,7 mM KCl, 0.1 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7 and the redox couple Fe(CN)63/Fe(CN)64 at 5 mM concentration. All reagents were of analytical grade and ultrapure water (resistance 18.2 MV cm 1) produced by a MilliporeMilli-Q system was used throughout. 2.2. Preparation of mixed self-assembled monolayers Evaporated gold (¨300 nm thickness) was deposited on silicon, using a titanium baselayer (30 nm thickness) as substrate. Before modification, the gold surface was cleaned in an ultrasonic bath for 10 min in acetone, dried under a dry N2 flow and then dipped for 1 min into ‘‘piranha solution’’ comprising 7 : 3 (v/v) 98% H2SO4 / 30% H2O2. The gold substrate was then rinsed 2 to 3 times with ultra-pure water and dried with a nitrogen flow. After cleaning, the gold electrodes were immediately immersed in an ethanol solution of 1 mM of MHDA and 0.1 mM of biotinyl-PE for 21 h at room temperature. After the formation of the mixed monolayer the substrate was rinsed 4 to 5 times with ethanol and dried under a N2 flow. 2.3. Blocking and interaction with biotinyl-PE-neutravidin Mixed self-assembled monolayer electrodes were immersed in 1 10 7 M non-specific IgG in PBS, pH 7, for 2 h to block the free space between the biotinylated species. The electrode was then thoroughly rinsed with PBS to remove excess of nonspecific IgG. Finally, the electrode was dipped into PBS containing 1 10 7 M neutravidin for 1 h 30min, and then rinsed with PBS buffer to remove non-specifically adsorbed neutravidin.
Neutravidin functionalised electrodes were immersed in 5 ml PBS at pH 7 with 1 108 M of biotinyl-anti-human haemoglobin at room temperature for 1 h, and then thoroughly rinsed with PBS to remove weakly absorbed antibodies. Finally, electrodes were subjected to various concentrations of antigen, and assayed in an electrochemical cell in order to measure the immunoreaction in real time.
2.6. Atomic force microscopy Atomic force microscopy (AFM) experiments were performed in air using PicoSMP microscopy with a 100 Am scanning head. The images were taken in tapping mode using a silicon pyramidal Si3N4 tips. The average resonance frequency of the tips was 75 kHz in air and the force constant about 3 Nm 1. All of the images presented in this paper were first-order flattened. 3. Results and discussion 3.1. Impedance spectroscopy Electrochemical impedance spectroscopy is a relatively new technique for characterising materials and surface interfaces, that has emerged with the development of instruments able to measure impedance as a function of frequency in the 0.5– 105 Hz range. The advantage of EIS over other electrochemical techniques is that only a small amplitude perturbations from steady state are needed, this makes it possible to treat the response theoretically by linearised or otherwise simplified current-potential characteristics. To fit the measured spectra to the impedance spectra model using real, rather than ideal elements, we replaced the ideal elements with the constant phase elements (CPE = Z, Eq. (1)) [15,16]: Z ¼ Kxa
ð1Þ
where K and a are experimental parameters (0 a 1). Three special cases depending on the a parameter can be considered:
324
S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
(A)
O
O O
NH
HN
O (R)
N
S
H
O
P O Na
H O
O
O O
(B)
16-mercaptohexadecanoic acid
Non-specific
Hemoglobin Anti-hemoglobin-Biotinyl
Neutravidin
Fig. 1. A) Chemical structure of biotinyl-PE, B) schematic showing the assembly of a mixed SAM based immunosensor (scale does not correspond to real size of species involved).
b
10000
6000
3.2. Electrochemical characteristics of mixed self-assembled monolayer
i
-Z (Ω)
8000
if a å 0, Z represents an ideal resistance and k = R; if a å 1 then Z corresponds to an ideal capacitor where K = 1 / C, and if a å 0.5 the circuit element is termed the Warburg impedance (W), where K = W, which is associated with a diffusion process. The a parameter is related to the area of defects and homogeneities in the surface layer, e.g., the SAM.
4000
a 2000
0 0
5000
10000
15000
20000
25000
Zr(Ω) Fig. 2. Complex impedance plots (Z r vs. Z i at 0 V vs. SCE in 0.1 mM PBS pH 7.0 solution) obtained for (a) bare gold electrode and (b) mixed SAMs in PBS. Spectra were obtained between 0.5 and 100 kHz.
Self-assembled monolayers consisting of long alkyl – thiols chains on gold have been shown to be stable in air, water and organic solvents at room temperature [17], whilst the biotin/ neutravidin couple has very high binding affinity. Therefore, it is possible to form a stable self-assembling system having potential application for the construction of biosensors. In this study, the mixed monolayer is composed of the thiol acid, MHDA, which possess a thiol group permitting its immobilisation on to gold surfaces, and a hydrophilic terminal carboxyl group. The final
S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
325
0.00020 0.00015
Current (A)
0.00010
a b
0.00005 0.00000 -0.00005
Fig. 3. Equivalent circuit used to model impedance data in PBS solution. -0.00010
stability of the mixed SAM layer is obtained through hydrophobic interaction between long alkyl (C16) chains. Fig. 1 shows the stages in the assembly of the mixed SAM based immunosensor, from initial mixed SAM formation, to the complete sensor which has bound the analyte (haemoglobin). Impedance measurements were performed in the frequency range from 0.5 to 105 Hz. Complex impedance plots of bare electrode (a) and mixed self-assembled monolayers (b) are shown in Fig. 2. The impedance spectra of bare gold electrode fit the theoretical profile and include a semi-circle region in the frequency range from 0.5 to 1 105 Hz. For the mixed selfassembled monolayers deposited on the gold electrode, the impedance spectra include a semi-circle region observed at high frequencies, corresponding to a change in the SAM structure, followed by a linear region characteristic of lower frequencies attributed to diffusion phenomenon. The respective compressed semi-circle diameters correspond to the membrane resistance at the electrode surface, and increase on the addition of self-assembled monolayers on the electrode surface. The impedance spectrum is commonly interpreted as a circuit model representative of the physicochemical processes involved. The present system can be modelled using the simple circuit shown in Fig. 3. Where R s is the solution resistance and the resistance of the electric contact, R m is the membrane resistance, C CPE is a double layer capacitance of constant phase element and W is the Warburg element. The values obtained for the elements R s, R m, C CPE, a and W for the bare gold electrode and mixed self-assembled monolayer are shown in Table 1. The values of the fractional coverage area of the mixed monolayer (h) can be calculated from the impedance diagrams using Eq. (2) [18]: h ¼ 1 Rm =R4m
ð2Þ
R *m
are the values of the membrane resistance where R m and derived from the impedance diagram of the bare gold electrode and mixed self-assembled monolayer, respectively. In our
-0.00015 -0.4
-0.2
0.0
0.2
0.4
0.6
Potential (V vs. SCE) Fig. 4. Cyclic voltammetry for bare gold and mixed SAM electrodes in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] in PBS pH 7.0. Electrodes were scanned at a rate of 50 mV/s ( a ), bare gold electrode; (b), mixed SAM covered electrode.
system the fractional coverage area was equal to 0.84. As can be seen, the high value of the Warburg impedance of the gold electrode with the covered SAM shows that the layer is not acting as a blocking layer but as a diffusion layer because of the low percentage of area coverage. Cyclic voltammetry experiments further confirmed that the mixed SAM was successfully formed on the gold surface. When the electrode surface was modified by addition of material, the electron transfer kinetics of Fe(CN)64/3 were perturbed. Fig. 4 shows the cyclic voltammograms of Fe(CN)64/3 at the bare gold electrode (curve a) and mixed SAM covered electrode (curve b). As shown in Fig. 4, the stepwise assembly of bare gold and mixed SAMs is accompanied by a decrease in the peak to peak separation between the cathodic and anodic waves of redox probe. This shows the formation of the mixed monolayer. 3.3. Detection of antibody and antigen – antibody interaction 3.3.1. Impedance spectroscopy As previously indicated, the schematic diagram for sensor fabrication and antibody binding is shown in Fig. 1B. Each step in the above procedure was followed by cyclic voltammetry which confirmed that the antibody was assembled on the gold surface. When the electrode surface was modified, the electron transfer kinetics of [Fe(CN)64/3] were perturbed. Fig. 5 shows the cyclic voltammetry in 5 mM of [Fe(CN)64/3] for mixed monolayer (curve a), neutravidin electrode (curve b) and
Table 1 Fitting values of the equivalent circuit elements for bare gold electrode, mixed SAM electrode layer, blocked with non-specific IgG, after binding of neutravidin and for the antibody R s (Vcm2) C CPE (Fcm 2) a R m (Vcm2) W (Vcm2)
Bare gold
Mixed self-assembled monolayer
IgG
Neutravidin
Antibody
247.2 3.369 10 6 0.942 2180 6800
231.1 6.68 10 7 0.93 13967 26699
236.5 6.06 10 7 0.94 9630 18868
252.1 5.25 10 7 0.96 5573 8329
236.6 5.3 10 7 0.96 4591 8081
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S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
0.00006
b c a
2000
0.00004
m
∆R (Ω)
Current (A)
1500 0.00002 0.00000 -0.00002
1000
500
-0.00004 0 -0.00006
0
-0.4
-0.2
0.0
0.2
0.4
0.6
Potential (V vs. SCE) Fig. 5. Cyclic voltametry of (a) mixed self-assembled monolayer/gold electrode, (b) neutravidin/IgG/mixed SAMs/ gold electrode and (c) antibodies/neutravidin/IgG/mixed SAMs/ gold electrode in the solution of 5 mM Fe(CN)4/3 in 0.1 mM PBS at pH 7.0. Scan rate, 50 mV/s. 6
antibody immobilized electrode (curve c). After addition of neutravidin the mixed self-assembled monolayer becomes less insulating and an increased current response was observed. This is could be attributed to the rearrangement in the structure of the SAMs, possibility due to multiple binding points occurring between neutravidin and the biotinyl surface. Adding on the biotinylated antibody to the neutravidin subsequently reduced the penetration of the redox pair and decreased the current response. Complex impedance plots of the sensors at the different stages of assembly (Fig. 1) are presented in Fig. 6. Using the equivalent circuit model described in Fig. 3, it is possible to fit the impedance spectra (Table 1). The specific interactions, for example between neutravidin and the biotin lipid in the SAM, between biotin tagged antibody and neutravidin, and between antibody and antigen (haemoglobin in this case) cause a decrease in the double layer capacitance constant phase a
10000
d
b
c
6000
i
-Z (Ω)
8000
4000 2000 0 0
5000
10000
15000
20000
25000
Zr(Ω) Fig. 6. Complex impedance plots ( Z r vs. Z i at 0V vs. SCE in 0.1 mM PBS pH 7.0 solution) of (a), mixed SAM; (b), mixed SAM blocked with non-specific IgG and (c), mixed SAM blocked with non-specific IgG and immobilized neutravidin and (d) antibody immobilized.
50
100
150
200
250
300
Concentration of haemoglobin (ng/ml) Fig. 7. Calibration plots of the variation of membrane resistance DR m with the concentration of antigen.
element and a decrease in membrane resistance. The decrease in the capacitance element is due to an increase in the thickness of the layer relating to Eq. (3): CCPE ¼ e0 ei A=dI
ð3Þ
where ( 0 is the vacuum dielectric constant (8.85 1014F/cm2), ( i is the dielectric constant of the layer i and A is the area of the surface. The membrane resistance decrease could be attributed to the change in the self-assembled structure. The electrode modified with antibody was dipped into an electrochemical cell and various concentrations of antigen were added. Data are presented in Fig. 7 which shows the membrane resistance difference (DR m) at the sensing interface with different concentrations of the antigen. The membrane resistance difference (DR m) is calculated following the equation: DRm ¼ RmðAbÞ RmðAbAgÞ As can be seen in Fig. 7, the plot shows saturation of the antibodies on the sensor surface with decreasing differences in the DR m value as the antibody pool reached saturation. A quasi-linear relationship between the membrane resistance values and the concentration of antigen held over the range from 10 to 100 ng/ml haemoglobin whilst the detection limit of the immunosensor was determined to be 20 ng/ml, or approximately 0.3 nM. From such a curve it is also possible to evaluate the dissociation constant Kd of the antigen/ antibody complex. As it corresponds to the haemoglobin concentration for half saturation Kd can be estimate around 5.8 10 11 M. 3.3.2. Atomic force microscopy Fig. 8A shows a modified surface achieved after immersion in 108 M of antibody at room temperature for 1 h. During the process of scanning no biological matter has been removed from the sample indicating that the antibody anti-human haemoglobin labelled with biotinyl was chemically bound to the freshly surface modified with neutravidin. However, all the images show a lack of surface homogeneity due to antibody aggrega-
S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
(A)
327
(B)
30nm
30nm
Fig. 8. Topography AFM images (scan are of 150 nm2); (A) Layer structure formed by anti-human hemoglobin labelled with biotinyl immobilised on the functionalised surface modified with neutravidin, (B) same as (A) after the injection of antigens.
tion. After injection of antigens, a different pattern respect to the first is observed (Fig. 8B), thus rendering the appearance of IgG due to the interaction between antigens and antibodies. 4. Conclusion Electrochemical impedance spectroscopy (EIS) was successfully used as a sensitive method for the detection of an antibody– antigen interaction. A novel mixed self-assembled monolayer was characterised by cyclic voltammetry and impedance spectroscopy. The biotinylated sensing-antibody was immobilized on the mixed monolayer using neutravidin and then reacted with antigen. Subsequently, the antibody – antigen interaction was monitored by the electrochemical impedance. Acknowledgments This work was financially supported by the European community under framework VI within project IMAGEMO (contract: QLK3-2001-02141). References [1] X. Cui, R. Pei, Z. Wang, F. Yang, Y. Ma, S. Dong, X. Yang, Biosensors and Bioelectronics 18 (2003) 59.
[2] F. Darain, D.S. Park, J.S. Park, Y.B. Shim, Biosensors and Bioelectronics 18 (2003) 773. [3] B. Corry, J. Uilk, C. Crawley, Analytica Chimica Acta 496 (2003) 103. [4] D. Piscevic, W. Knoll, M.J. Tarlov, Supramolecular Science 2 (1995) 99. [5] S.J. Linda, K.E. Nelson, C.T. Campbell, P.S. Stayton, S.S. Yee, V. Pe´rezLuna, G.P. Lo´pez, Sensors and Actuators B 54 (2000) 137. [6] M.B. Gonza´lez-Garcia, C. Fernandez-Sanchez, A. Costa-Garcia, Biosensors and Bioelectronics 15 (2000) 315. [7] J.R. Sousa, M.M.V. Parente, I.C.N. Diogenes, L.G.F. Lopes, P.L. Neto, M.L.A. Temperini, Al.A. Batista, I.S. Moreira, Journal of Electroanalytical Chemistry 566 (2004) 443. [8] S. Lee, S.S. Young, R. Colorado Jr., R.L. Guenard, T.R. Lee, S.S. Perry, Langmuir 16 (2000) 2220. [9] R.P. Janek, W.R. Fawcett, A. Ulman, Langmuir 14 (1998) 3011. [10] U.S. Kumar, V. Lakshminarayanan, Journal of Electroanalytical Chemistry 565 (2004) 343. [11] P.J. Richard, W.R. Fawcett, U. Abraham, Journal of Physical Chemistry. B 101 (1997) 8550. [12] V. Molinero, J.C. Ernesto, Journal of Electroanalytical Chemistry 445 (1998) 17. [13] P.S. Germain, W.G. Pell, B.E. Conway, Electrochimica Acta 49 (2004) 1775. [14] X. Cui, D. Jiang, P. Li, J. Diao, R. Tong, X. Wang, Electroanalytical Chemistry 470 (1999) 9. [15] I. Navratilova, P. Skladal, Bioelectrochemistry 62 (2004) 11. [16] J. Benavente, J.M. Garcia, R. Riley, A.E. Lozano, J. Abajo, Membrane Science 175 (2000) 43. [17] J.L. Tan, J. Tien, C.S. Chen, Langmuir 18 (2002) 519. [18] J.R. Sousa, Ma.M.V. Parente, I.C.No. Diogenes, L.G.F. Lopes, P.Li. Neto, M.L.A. Temperini, Al.Az. Batista, I.S. Moreira, Journal of Electroanalytical Chemistry 566 (2004) 443.
An immunosensor for haemoglobin based on impedimetric properties of a new mixed self-assembled monolayer S. Hleli a,b, C. Martelet a,*, A. Abdelghani b, F. Bessueille c, A. Errachid c, J. Samitier c, H.C.W. Hays d, P.A. Millner d, N. Burais a, N. Jaffrezic-Renault a b
a Centre de Ge´nie Electrique de Lyon, CEGELY, ECL, 69134 Ecully cedex, France Unite´ de recherche de physique des semiconducteurs et capteurs, IPEST, La Marsa, Tunisia c Laboratory of NanoBioEngineering, Barcelona Science Park, Barcelona 08028, Spain d School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
Available online 7 December 2005
Abstract A novel impedimetric immunosensor for the detection of haemoglobin has been developed by mixed self-assembled monolayers on Au. First, a mixed self-assembled monolayer (SAMs) consisting of 1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotinyl-PE) and 16mercaptohexadecanoic acid (MHDA) on gold electrodes was studied. The conformational properties of the SAMs were characterised by cyclic voltammetry and impedance spectroscopy. After blocking non-specific binding sites in the mixed monolayer using non-specific IgG, neutravidin was used to bind to biotinyl sites present in the mixed monolayer. Finally, biotinylated anti-haemoglobin IgG was immobilised to the tethered neutravidin. The membrane resistance R m, obtained from the assembly, decreased gradually after the addition of non-specific IgG, neutravidin and anti-haemoglobin to the monolayer. This decrease could be attributed to a rearrangement in the structure of the SAMs. The detection of antibody – antigen reaction demonstrates that the potentiometric immunosensor exhibited high sensitivity and a detection limit of 20 ng/ml (approximately 0.3 nM). D 2005 Elsevier B.V. All rights reserved. Keywords: Human haemoglobin; Biotinyl-PE; Neutravidin; Mixed self-assembled monolayers; Impedance spectroscopy; AFM; Biosensors
1. Introduction The development of numerous immunochemical methods for the measurement of antibody –antigen reactions continues to be the subject of considerable research and development effort [1 – 3]. In the last two decades, many reports have been published on the use of immunosensors for a wide range of applications in food, environment, pharmaceutical chemistry and clinical diagnostics. Immunosensors are based on the use of an antibody that reacts specifically with the substance (antigen) to be tested. Therefore, one of the most important points in the design of an immunosensor is the choice of immobilization method. Recently, self-assembled monolayers (SAMs) generated by the adsorption of alkanethiols on gold have been the subject of extensive research due to their simple preparation and special characteristics. One of the best understood functionalised self* Corresponding author. Tel.: +33 472186248; fax: +33 478433717. E-mail address: [email protected] (C. Martelet). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.10.054
assembled monolayer systems is that based on biotin/streptavidin to attach a sensor biomolecule, which is usually an enzyme or an antibody [4 – 7]. Streptavidin has an extremely high binding affinity for biotin (Ka¨1015M) [8,9] and the streptavidin molecule is a homotetramer with each subunit having a single biotin binding site. Therefore, it can be used as a linker between two biotin tagged components, such as the surface of an electrode and a biotin tagged protein. Among the various transduction techniques, electrochemical impedance spectroscopy (EIS) has previously been investigated to study the selective binding of streptavidin to avidinterminated alkanethiols and disulfide or sulfide containing molecules self-assembled on gold [10 – 13]. EIS is a sensitive technique, which monitors the electrical response of the system studied after application of a periodic small amplitude AC signal. Analysis of the system response provides information concerning the interface and reactions occurring at it [14,15]. EIS has been widely used by several workers to characterise SAM quality [16].
S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
323
In this work, IgG directed against human haemoglobin was immobilised on a mixed self-assembled layer with neutravidin and faradaic impedance spectroscopy was applied as the electrochemical sensing method. A novel mixed self-assembled monolayer consisting of biotinyl-PE and MHDA was examined using electrochemical impedance spectroscopy and cyclic voltammetry. The neutravidin/biotin interaction and conformational effects induced within the SAM were detected using the impedance spectroscopy.
2.4. Immunoassay of haemoglobin
2. Experimental
2.5. Impedance spectroscopy
2.1. Material
The measurement set-up for impedance consisted of a classical three-electrode system, where the modified gold electrode (0.21 cm2) was used as working electrode, a platinum strip (0.54 cm2) as a counter electrode and a saturated calomel electrode (SCE) as reference electrode. The impedance analysis was performed with the Voltalab 80 impedance analyser in the frequency range 0.5 Hz – 100 kHz, using a modulation voltage of 10 mV. During measurements the potential was kept at zero volts. The impedance measurements and cyclic voltametry measurements were performed in the presence of a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] (1 : 1) mixture as redox probe in PBS. The Zview modelling programme (Scriber and associates, Charlottesville, VA) was used. All electrochemical measurements were carried out at room temperature and in a faraday cage.
Immunoglobulin G (IgG) against human haemoglobin was from bethyl and was biotinylated using N-(+)-biotinyl-6aminocaproic acid N-succinimidyl ester according to manufacturers instructions. Human haemoglobin and non-immune goat IgG were purchased from Sigma-Aldrich whilst neutravidin was obtained from Pierce. The affinity phospholipid dipalmitoyl-snglycero-3-phospho-ethanolamine-N-(biotinyl) (biotinyl-PE) and 16-mercaptohexadecanoic acid, for the formation of mixed monolayers were purchased from Avanti polar lipids and SigmaAldrich, respectively. The buffer solution used for all experiments was phosphate buffered saline (PBS) containing 140 mM NaCl, 2,7 mM KCl, 0.1 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7 and the redox couple Fe(CN)63/Fe(CN)64 at 5 mM concentration. All reagents were of analytical grade and ultrapure water (resistance 18.2 MV cm 1) produced by a MilliporeMilli-Q system was used throughout. 2.2. Preparation of mixed self-assembled monolayers Evaporated gold (¨300 nm thickness) was deposited on silicon, using a titanium baselayer (30 nm thickness) as substrate. Before modification, the gold surface was cleaned in an ultrasonic bath for 10 min in acetone, dried under a dry N2 flow and then dipped for 1 min into ‘‘piranha solution’’ comprising 7 : 3 (v/v) 98% H2SO4 / 30% H2O2. The gold substrate was then rinsed 2 to 3 times with ultra-pure water and dried with a nitrogen flow. After cleaning, the gold electrodes were immediately immersed in an ethanol solution of 1 mM of MHDA and 0.1 mM of biotinyl-PE for 21 h at room temperature. After the formation of the mixed monolayer the substrate was rinsed 4 to 5 times with ethanol and dried under a N2 flow. 2.3. Blocking and interaction with biotinyl-PE-neutravidin Mixed self-assembled monolayer electrodes were immersed in 1 10 7 M non-specific IgG in PBS, pH 7, for 2 h to block the free space between the biotinylated species. The electrode was then thoroughly rinsed with PBS to remove excess of nonspecific IgG. Finally, the electrode was dipped into PBS containing 1 10 7 M neutravidin for 1 h 30min, and then rinsed with PBS buffer to remove non-specifically adsorbed neutravidin.
Neutravidin functionalised electrodes were immersed in 5 ml PBS at pH 7 with 1 108 M of biotinyl-anti-human haemoglobin at room temperature for 1 h, and then thoroughly rinsed with PBS to remove weakly absorbed antibodies. Finally, electrodes were subjected to various concentrations of antigen, and assayed in an electrochemical cell in order to measure the immunoreaction in real time.
2.6. Atomic force microscopy Atomic force microscopy (AFM) experiments were performed in air using PicoSMP microscopy with a 100 Am scanning head. The images were taken in tapping mode using a silicon pyramidal Si3N4 tips. The average resonance frequency of the tips was 75 kHz in air and the force constant about 3 Nm 1. All of the images presented in this paper were first-order flattened. 3. Results and discussion 3.1. Impedance spectroscopy Electrochemical impedance spectroscopy is a relatively new technique for characterising materials and surface interfaces, that has emerged with the development of instruments able to measure impedance as a function of frequency in the 0.5– 105 Hz range. The advantage of EIS over other electrochemical techniques is that only a small amplitude perturbations from steady state are needed, this makes it possible to treat the response theoretically by linearised or otherwise simplified current-potential characteristics. To fit the measured spectra to the impedance spectra model using real, rather than ideal elements, we replaced the ideal elements with the constant phase elements (CPE = Z, Eq. (1)) [15,16]: Z ¼ Kxa
ð1Þ
where K and a are experimental parameters (0 a 1). Three special cases depending on the a parameter can be considered:
324
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(A)
O
O O
NH
HN
O (R)
N
S
H
O
P O Na
H O
O
O O
(B)
16-mercaptohexadecanoic acid
Non-specific
Hemoglobin Anti-hemoglobin-Biotinyl
Neutravidin
Fig. 1. A) Chemical structure of biotinyl-PE, B) schematic showing the assembly of a mixed SAM based immunosensor (scale does not correspond to real size of species involved).
b
10000
6000
3.2. Electrochemical characteristics of mixed self-assembled monolayer
i
-Z (Ω)
8000
if a å 0, Z represents an ideal resistance and k = R; if a å 1 then Z corresponds to an ideal capacitor where K = 1 / C, and if a å 0.5 the circuit element is termed the Warburg impedance (W), where K = W, which is associated with a diffusion process. The a parameter is related to the area of defects and homogeneities in the surface layer, e.g., the SAM.
4000
a 2000
0 0
5000
10000
15000
20000
25000
Zr(Ω) Fig. 2. Complex impedance plots (Z r vs. Z i at 0 V vs. SCE in 0.1 mM PBS pH 7.0 solution) obtained for (a) bare gold electrode and (b) mixed SAMs in PBS. Spectra were obtained between 0.5 and 100 kHz.
Self-assembled monolayers consisting of long alkyl – thiols chains on gold have been shown to be stable in air, water and organic solvents at room temperature [17], whilst the biotin/ neutravidin couple has very high binding affinity. Therefore, it is possible to form a stable self-assembling system having potential application for the construction of biosensors. In this study, the mixed monolayer is composed of the thiol acid, MHDA, which possess a thiol group permitting its immobilisation on to gold surfaces, and a hydrophilic terminal carboxyl group. The final
S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
325
0.00020 0.00015
Current (A)
0.00010
a b
0.00005 0.00000 -0.00005
Fig. 3. Equivalent circuit used to model impedance data in PBS solution. -0.00010
stability of the mixed SAM layer is obtained through hydrophobic interaction between long alkyl (C16) chains. Fig. 1 shows the stages in the assembly of the mixed SAM based immunosensor, from initial mixed SAM formation, to the complete sensor which has bound the analyte (haemoglobin). Impedance measurements were performed in the frequency range from 0.5 to 105 Hz. Complex impedance plots of bare electrode (a) and mixed self-assembled monolayers (b) are shown in Fig. 2. The impedance spectra of bare gold electrode fit the theoretical profile and include a semi-circle region in the frequency range from 0.5 to 1 105 Hz. For the mixed selfassembled monolayers deposited on the gold electrode, the impedance spectra include a semi-circle region observed at high frequencies, corresponding to a change in the SAM structure, followed by a linear region characteristic of lower frequencies attributed to diffusion phenomenon. The respective compressed semi-circle diameters correspond to the membrane resistance at the electrode surface, and increase on the addition of self-assembled monolayers on the electrode surface. The impedance spectrum is commonly interpreted as a circuit model representative of the physicochemical processes involved. The present system can be modelled using the simple circuit shown in Fig. 3. Where R s is the solution resistance and the resistance of the electric contact, R m is the membrane resistance, C CPE is a double layer capacitance of constant phase element and W is the Warburg element. The values obtained for the elements R s, R m, C CPE, a and W for the bare gold electrode and mixed self-assembled monolayer are shown in Table 1. The values of the fractional coverage area of the mixed monolayer (h) can be calculated from the impedance diagrams using Eq. (2) [18]: h ¼ 1 Rm =R4m
ð2Þ
R *m
are the values of the membrane resistance where R m and derived from the impedance diagram of the bare gold electrode and mixed self-assembled monolayer, respectively. In our
-0.00015 -0.4
-0.2
0.0
0.2
0.4
0.6
Potential (V vs. SCE) Fig. 4. Cyclic voltammetry for bare gold and mixed SAM electrodes in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] in PBS pH 7.0. Electrodes were scanned at a rate of 50 mV/s ( a ), bare gold electrode; (b), mixed SAM covered electrode.
system the fractional coverage area was equal to 0.84. As can be seen, the high value of the Warburg impedance of the gold electrode with the covered SAM shows that the layer is not acting as a blocking layer but as a diffusion layer because of the low percentage of area coverage. Cyclic voltammetry experiments further confirmed that the mixed SAM was successfully formed on the gold surface. When the electrode surface was modified by addition of material, the electron transfer kinetics of Fe(CN)64/3 were perturbed. Fig. 4 shows the cyclic voltammograms of Fe(CN)64/3 at the bare gold electrode (curve a) and mixed SAM covered electrode (curve b). As shown in Fig. 4, the stepwise assembly of bare gold and mixed SAMs is accompanied by a decrease in the peak to peak separation between the cathodic and anodic waves of redox probe. This shows the formation of the mixed monolayer. 3.3. Detection of antibody and antigen – antibody interaction 3.3.1. Impedance spectroscopy As previously indicated, the schematic diagram for sensor fabrication and antibody binding is shown in Fig. 1B. Each step in the above procedure was followed by cyclic voltammetry which confirmed that the antibody was assembled on the gold surface. When the electrode surface was modified, the electron transfer kinetics of [Fe(CN)64/3] were perturbed. Fig. 5 shows the cyclic voltammetry in 5 mM of [Fe(CN)64/3] for mixed monolayer (curve a), neutravidin electrode (curve b) and
Table 1 Fitting values of the equivalent circuit elements for bare gold electrode, mixed SAM electrode layer, blocked with non-specific IgG, after binding of neutravidin and for the antibody R s (Vcm2) C CPE (Fcm 2) a R m (Vcm2) W (Vcm2)
Bare gold
Mixed self-assembled monolayer
IgG
Neutravidin
Antibody
247.2 3.369 10 6 0.942 2180 6800
231.1 6.68 10 7 0.93 13967 26699
236.5 6.06 10 7 0.94 9630 18868
252.1 5.25 10 7 0.96 5573 8329
236.6 5.3 10 7 0.96 4591 8081
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0.00006
b c a
2000
0.00004
m
∆R (Ω)
Current (A)
1500 0.00002 0.00000 -0.00002
1000
500
-0.00004 0 -0.00006
0
-0.4
-0.2
0.0
0.2
0.4
0.6
Potential (V vs. SCE) Fig. 5. Cyclic voltametry of (a) mixed self-assembled monolayer/gold electrode, (b) neutravidin/IgG/mixed SAMs/ gold electrode and (c) antibodies/neutravidin/IgG/mixed SAMs/ gold electrode in the solution of 5 mM Fe(CN)4/3 in 0.1 mM PBS at pH 7.0. Scan rate, 50 mV/s. 6
antibody immobilized electrode (curve c). After addition of neutravidin the mixed self-assembled monolayer becomes less insulating and an increased current response was observed. This is could be attributed to the rearrangement in the structure of the SAMs, possibility due to multiple binding points occurring between neutravidin and the biotinyl surface. Adding on the biotinylated antibody to the neutravidin subsequently reduced the penetration of the redox pair and decreased the current response. Complex impedance plots of the sensors at the different stages of assembly (Fig. 1) are presented in Fig. 6. Using the equivalent circuit model described in Fig. 3, it is possible to fit the impedance spectra (Table 1). The specific interactions, for example between neutravidin and the biotin lipid in the SAM, between biotin tagged antibody and neutravidin, and between antibody and antigen (haemoglobin in this case) cause a decrease in the double layer capacitance constant phase a
10000
d
b
c
6000
i
-Z (Ω)
8000
4000 2000 0 0
5000
10000
15000
20000
25000
Zr(Ω) Fig. 6. Complex impedance plots ( Z r vs. Z i at 0V vs. SCE in 0.1 mM PBS pH 7.0 solution) of (a), mixed SAM; (b), mixed SAM blocked with non-specific IgG and (c), mixed SAM blocked with non-specific IgG and immobilized neutravidin and (d) antibody immobilized.
50
100
150
200
250
300
Concentration of haemoglobin (ng/ml) Fig. 7. Calibration plots of the variation of membrane resistance DR m with the concentration of antigen.
element and a decrease in membrane resistance. The decrease in the capacitance element is due to an increase in the thickness of the layer relating to Eq. (3): CCPE ¼ e0 ei A=dI
ð3Þ
where ( 0 is the vacuum dielectric constant (8.85 1014F/cm2), ( i is the dielectric constant of the layer i and A is the area of the surface. The membrane resistance decrease could be attributed to the change in the self-assembled structure. The electrode modified with antibody was dipped into an electrochemical cell and various concentrations of antigen were added. Data are presented in Fig. 7 which shows the membrane resistance difference (DR m) at the sensing interface with different concentrations of the antigen. The membrane resistance difference (DR m) is calculated following the equation: DRm ¼ RmðAbÞ RmðAbAgÞ As can be seen in Fig. 7, the plot shows saturation of the antibodies on the sensor surface with decreasing differences in the DR m value as the antibody pool reached saturation. A quasi-linear relationship between the membrane resistance values and the concentration of antigen held over the range from 10 to 100 ng/ml haemoglobin whilst the detection limit of the immunosensor was determined to be 20 ng/ml, or approximately 0.3 nM. From such a curve it is also possible to evaluate the dissociation constant Kd of the antigen/ antibody complex. As it corresponds to the haemoglobin concentration for half saturation Kd can be estimate around 5.8 10 11 M. 3.3.2. Atomic force microscopy Fig. 8A shows a modified surface achieved after immersion in 108 M of antibody at room temperature for 1 h. During the process of scanning no biological matter has been removed from the sample indicating that the antibody anti-human haemoglobin labelled with biotinyl was chemically bound to the freshly surface modified with neutravidin. However, all the images show a lack of surface homogeneity due to antibody aggrega-
S. Hleli et al. / Materials Science and Engineering C 26 (2006) 322 – 327
(A)
327
(B)
30nm
30nm
Fig. 8. Topography AFM images (scan are of 150 nm2); (A) Layer structure formed by anti-human hemoglobin labelled with biotinyl immobilised on the functionalised surface modified with neutravidin, (B) same as (A) after the injection of antigens.
tion. After injection of antigens, a different pattern respect to the first is observed (Fig. 8B), thus rendering the appearance of IgG due to the interaction between antigens and antibodies. 4. Conclusion Electrochemical impedance spectroscopy (EIS) was successfully used as a sensitive method for the detection of an antibody– antigen interaction. A novel mixed self-assembled monolayer was characterised by cyclic voltammetry and impedance spectroscopy. The biotinylated sensing-antibody was immobilized on the mixed monolayer using neutravidin and then reacted with antigen. Subsequently, the antibody – antigen interaction was monitored by the electrochemical impedance. Acknowledgments This work was financially supported by the European community under framework VI within project IMAGEMO (contract: QLK3-2001-02141). References [1] X. Cui, R. Pei, Z. Wang, F. Yang, Y. Ma, S. Dong, X. Yang, Biosensors and Bioelectronics 18 (2003) 59.
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