Anal. Chem. 1998, 70, 5072-5078
Immunoelectrodes in Protein Detection: Comparison between Glassy Carbon and a Semimetallic Ni/P Thin Film as Binding Support. Biological Applications Pascal Darbon,† Vincent Michel,† Franc¸ ois Math,*,† Herve´ Giorgi,‡ and Francis Machizaud§
Laboratoire de Neurosciences, UFR Sciences et Techniques, Universite´ de Franche-Comte´ , 1 place Leclerc, 25030 Besanc¸ on, France, Laboratoire de Biophysique, Faculte´ de Medecine, Universite´ de Franche-Comte´ , place St Jacques, 25030 Besanc¸ on, France, and Laboratoire Sciences et Ge´ nie des Mate´ riaux Me´ talliques, INPL CNRS, Universite´ de Nancy I, 54000 Nancy, France
Detection of very low amounts of proteins, in real time, in situ or in vitro, is expected by most researchers in biology. Voltammetric or amperometric techniques can detect only a restricted range of chemicals such as catechols or indols. Moreover, since our aim was to detect neuropeptides in the brain tissues, in vivo or in vitro, the potential which must be applied for oxidization of these substances was too high (more than 200 mV) and compro-
mised further measurements, because it progressively led to the alteration of an indefinite quantity of chemicals. Some other techniques are available, such as microdialysis or push-pull cannula, but they may induce extensive tissue or cell lesions and/ or need for further tissue preparation for analysis. To avoid these various problems, an antiboby-coated probe has been developed, which was previously named an immunoelectrode.1 This immunoelectrode detected antigen amounts as low as 0.1 pmol, using the appropriate specific antibody by way of electric charge displacements generated by the antigen/antibody (Ag/Ab) complex formation. Detected antigen concentrations were in the range of 10-7-10-15 M. Antibodies must be firmly bound to the glassy carbon fibers carrying the currents induced by Ag/Ab complex formation; carbon fibers are connected to a very low-noise and sensitive amplifier because Ag/Ab binding and associated ions displacements generated currents in the range of 1 nA. This detection method has already been successfully used for detection of somatostatin-like somatic exocytosis on snail visceral ganglion neurons either due to the spontaneous activity of cultured neurons2 or after in situ electrical stimulation of isolated neurons.3 We have previously reported a manufacturing method for reproductible carbon fiber microelectrodes.1 Our goal in this work was first to improve the preparation steps of immunoelectrodes. Particular attention must be paid to the setup of the electrode manufacturing since carbon fibers or any other appropriate materials, conductive enough to be an electrode, are the basic support for antibody binding and have to carry the electrical charge toward the amplifier. Their properties must allow uniform antibody binding without any failure (e.g., gas bubbles leaving uncoated area), because any default in the coating density can generate noise caused by unspecific bound proteins. Because of the very tiny current density, we have searched for higher conductivity in order to collect larger current flows, minimizing leakage in the medium. Two types have been tested: (1) the medium conductivity of carbon fibers with several thin fibers in
* To whom correspondence should be addressed. Fax: (33) 03 81 66 57 54. E-mail:
[email protected]. † Laboratoire de Neurosciences, Universite ´ de Franche-Comte´. ‡ Laboratoire de Biophysique, Universite ´ de Franche-Comte´. § Universite ´ de Nancy I.
(1) Math, F.; Marianneau, G. J. Neurosci. Methods 1994, 52, 149-51. (2) Pellegrini, E.; Dadkah-Monnier, Z.; Bride, M.; Math, F. Ann. Int. Conf. IEEE Eng. Med. Biol. 1992, 239-42. (3) Darbon, P.; Monnier, Z.; Bride, M.; Crest, M.; Gola, M.; Marianneau, G.; Math, F. J. Neurosci. Methods 1996, 67, 197-201.
Though immunoelectrodes can allow direct detection of very low protein amounts (about 0.1 pmol) in vitro and in vivo, they are not yet widely used because they need quality improvement. Based on a few works devoted to the basic electrochemical phenomenon occurring when antibobies are linked onto a solid support and during antigen/antibody complex formation, we have coated two different supports with antibodies: the classical glassy carbon fiber or an epoxy plate covered with an amorphous semimetallic (nickel/phosphorus) thin film obtained by means of an electrochemical deposit. The antibody/ antigen complex formation induces direct and/or indirect ionic movements and a current flow through the conductive support toward a very low-noise and high-sensitivity preamplifier stage in an I/V configuration. The proposed electrochemical treatment (hydrophilization), applied to both carbon and Ni/P electrodes, improves antibody binding and reliability of the response to antigens. The Ni/P probes present several advantages when compared to carbon fiber: better conductivity, possibility of surface quality control, and semimetallic nature, making them unbreakable. Several applications were proposed: somatostatin-14 detection with both carbon fiber and Ni/P plate electrodes, and histamine detection in simple and complex fluid media. Dose-response curves and analysis of the results lead us to conclude that the obtained currents are directly related to the quantity of antigen.
5072 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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a bundle, or with only one larger fiber, and (2) the conductivity of another new material which acts like carbon fiber toward antibodies and which conducts the current perfectly. This last material is thought to progressively replace carbon fiber because it extends the use of immunoelectrodes to a large number of applications, mostly because it is an unbreakable and very strong material. This material is an amorphous semimetallic deposit on an epoxy plate. Finally, we propose a simplified theoretical approach to the process that might occur when immunoelectrodes convert antibody-antigen complex formation into measurable current. EXPERIMENTAL SECTION Glassy Carbon Fiber Electrode Manufacture. Glassy carbon fibers are already widely used in electrophysiology, in amperometry, and in enzyme-coated electrodes. The carbon fibers used were made of ex-viscose (Toray Inc., Japan), 5 µm in diameter. Two kinds of carbon fibers electrodes were manufactured: single-fiber electrodes and multifibers electrodes made of a bundle of 5-10 fibers. The free end of the carbon fiber bundle was brought near one end of a copper wire (0.1 mm in diameter), to which it was glued using a silver-containing conductive resin (Epotecny, H20E). This process was stereomicroscopically controlled in order to set fibers firmly in line with the copper wire and to check on the gluing quality. After the electrode was dried at 100 °C, its resistance was measured by immersing its tip in mercury; values around 100 Ω were usually obtained. In some tests, we used a single-fiber electrode with a larger diameter, 30 µm (supplied from WPI, UK), instead of a multifiber electrode. Ni/P Plate Electrode Manufacture. Ni/P has been previously developed for some metallic limb prothesis overlying because such material was thought to evoke no tissue reaction. Moreover, it has semimetallic properties (e.g., a good electrical conductivity and low current noise in saline solution), and this material is less breakable, when deposited on a thin tungsten wire, than carbon fibers of the same diameter. In our first tests, we have used large copper-covered epoxy plates (20 mm × 5 mm) cut from electronic plate supplies. Preparation of Ni/P plates included removing grease from the copper surface and polishing by a treatment with a very fine diamond powder. An amorphous nickel/phosphorus metallic thin film of about 10 µm thickness was layered by electrolysis. A 10-V potential was applied during 2 h through two similar plates immersed in a sodium lipophosphite solution containing 19% Ni, pH 2-3. Current density was adjusted to 17 mA/cm2. Electrolytic parameters and alloy nature were modified until the best thin-film adhesion and surface homogeneity were obtained.4 Inappropriate equilibrium between Ni and P in the bath could produce a heterogeneous plate surface with microfractures due to random crystallization. The best homogeneous thin films were obtained by two or three successive electrolytic deposits, reaching a final thickness of 8-10 µm. Optimization of current and pH parameters during each electrolytic step was done by control of the surface under metallographic microscopy (Figure 1). Using such a process, we obtained electrodes devoid of microfracture, having a good adherence over all the surface and an absence or a low density of hydrogen bubbles. Surface aspect was further observed under scanning (4) Math, F.; Machizaud, F. Licence No. 45540-221195, 1995.
Figure 1. Metallographic microphotography in polarized light of semi-metallic Ni/P electrode. (A) (×26) Left epoxy plate with copper covering, right epoxy plate copper covered with amorphous metallic Ni/P coating. (B) (×205) Amorphous metallic Ni/P coating on epoxy copper-coated plate. Ni/P plate electrodes with homogeneous surface devoid of microfracture and an absence or a low density of hydrogen bubbles.
tunneling microscopy and with X-analysis microscopy (not shown). Scanning tunneling was used to control the surface appearance before any treatment. The X-analysis microscopy was used to control the presence of antibody after adsorption with a goldlabeled antibody. Electrochemical Treatment. Before the electrode was coated with antibodies, an electrochemical treatment was necessary to bind antibodies on the support surface. This electrochemical treatment included several steps. In the first step, electrodes were immersed in PBS (phosphate-buffered saline, 0.14 M NaCl, 2 mM KH2PO4, 6.5 mM Na2HPO4, pH 7.4) or in snail Ringer saline (80 mM NaCl, 4 mM KCl, 8 mM CaCl2, 8 mM MgCl2, 5 mM glucose, and 5 mM Tris-HCl, pH 7.6). While electrodes were immersed, we applied a triangular voltage waveform between -1 and +1 V (70 Hz) through one electrode against the other electrode or an anode made of a platinum wire. The electrode was then plunged in a bath of KMnO4 solution at saturation. Finally, the electrode was put into a diluted antibody solution (1/ 10) for at least 1 h to adsorb antibodies on the activated electrode surface. Amplifier Description. Antigen binding onto antibodies induced a response picked up by the immunoelectrode. The response was detected by the amplifier as a transient current in Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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Figure 2. Comparison of surface molecular arrangement of (A) the structure of carbon network (from PUTNIS8) made up of layers in which the carbon atoms lie at the corner of a hexagonal mesh and of (B) the Ni/P network which consists of a pseudo-randomized network containing prismatic structures linked together by two Ni atoms.
a range of pico- to nanoamperes. Amplification was achieved with a preamplifier stage in an I/V configuration, the inputs of which consisted in two parallel AD 549 operational amplifiers (analog devices) with extremely low bias current (30 fA) and a very high input impedance (1015 Ω). This electronic stage was used as a low-noise current-to-voltage converter (1 mV/1 nA). The detected transient currents could reach intensities which were in the nanoampere range, and their detection was improved by using two similar electrodes for differential measurements. Though both electrodes were electrochemically treated, we coated only one electrode with antibody. Differential measurements allowed in improvement of the signal-to-noise ratio of the recorded potentials. But the use of a constant-potential reference electrode (such as platinum or silver-silver chloride wires) could also yield good quantitative results after an appropriate low-frequency filtering. Molecular Modeling of the Somatostatin Molecule. The molecular modeling was done with Insight II on a SiliconGraphics computer from the data on the primary structure,5 on the tertiary structure,6,7 and on the SYBYL database done by S. J. Hocart (Peptide Research Laboratories, Tulane University, New Orleans, LA, available at http://peptide.med.tulane.edu). We have looked for the lower energy conformer at 300 K and with a dielectric constant ) 80. RESULTS AND DISCUSSION Principles and Basis of the Electrochemical Treatment. A strong and homogeneous antibody coating of the electrode surface is required in order to carry the charge flow produced during Ag/Ab complex formation to the preamplifier stage via the electrode as well as to avoid any other protein binding. This coating depended on the electrochemical treatment performed for a suitable binding of antibodies to the electrode surface. The electrochemical treatment has been improved on the carbon fiber and then applied to Ni/P plate electrodes. The appropriate electrochemical treatment consists of a hydrophilization, i.e., carboxyl groups formation, of the carbon surface described by Malmsten et al.8 But we have also considered the antibody binding principles as described by Quash et al.,9 who coupled antibodies to latex spheres using a similar hydrophilization method. Quash et al. generated aldehyde groups, by the action of periodate on carbohydrate, which are situated 5074 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
primarily on the CH2 region of the Fc portion of the antibody molecule. Simultaneously, they inserted sidearms onto latex spheres with an acid hydrazide end group. Then IgGs were coupled, via the aldehydes generated, to acid hydrazide groups at the end of the sidearm attached to latex spheres. According to Quash et al.9 and to the observations of Malmstem and Holmberg (Institut for Surface Chemistry, Stockolm, Sweden) on hydrophilized carbon fiber electrodes, and to our own tests, we have retained the electrochemical treatment described in the Experimental Section. An electrical treatment was applied in order to generate carboxyl groups at the carbon surface. Carboxyl groups appeared, as shown by Pantano and Kuhr,10 as the result of the breaking of the bond between two carbon atom at the carbon fiber surface. A KMnO4 bath was used to eliminate gaseous O2 in the electrode network. The electrochemical treatment applied on the Ni/P plate electrode has the same characteristics as that on a glassy carbon fiber electrode. Carbon and Ni/P atomic networks are compared in Figure 2. It can be seen that the Ni/P surface consists of a pseudo-randomized network containing prismatic structures linked together by two Ni atoms. A triangular voltage waveform (-1/ +1 V; 70 Hz) induced NiOH groups in the Ni/P prismatic arrangement. During this step, atomic arrangements occurred in the Ni/P network which made the Ni/P plate surface appropriate for antigen binding. Despite the differences existing between graphite and Ni/P networks, it appeared that the properties of this structure are suitable for antibodies binding. Antibodies could be bound to Ni atoms through NiOH groups formed during electrochemical treatment. The generated NiOH groups inside the nickel/phosphorus covering network acted like the carboxyl groups which were formed in the graphite network on carbon (5) Gustausson, S.; Lundquist, G. Biochem. Biophys. Res. Commun. 1978, 82, 1229-42. (6) Han, S. L.; Rivier, J. E.; Scheraga, H. A. Int. J. Pept. Protein Res. 1980, 15, 355-64. (7) Strnad, J.; Hadcock, J. R. Biochem. Biophys. Res. Commun. 1995, 216, 91321. (8) Malmsten, M.; Lassen, B.; Holmberg, K.; Thomas, V.; Quash, G. J. Colloid Interface Sci. 1996, 177, 70-8. (9) Quash, G. A.; Thomas, V.; Ogier, G.; El Alaoui, S.; Delcros, J. G.; Ripoll, H.; Roch, A. M.; Legastelois, S.; Gibert, R.; Ripoll, J. P. Covalently modified antigens and antibodies in diagnosis and therapy; Dekker: New York, 1989; Chapter 8. (10) Pantano, P.; Kuhr, W. G. Anal. Chem. 1993, 65, 623-30.
Figure 3. Scanning electron microphotography (white bar ) 1 µm) showing the three successive surface states of the amorphous metallic Ni/P coating without electrochemical treatment (A), after electrochemical treatment (hydrophilization) as described above (B), and after electrochemical treatment followed by antibody coating and antigen addition showing the Ag/Ab complex network (C).
fibers. The next electrochemical treatment steps were identical to those mentioned above. Scanning electron microscopy (Figure 3) showed three successive surface states of the amorphous metallic Ni/P coating without electrochemical treatment (A), after electrochemical treatment (hydrophilization) as described above (B), and after electrochemical treatment followed by antibody coating and antigen addition showing the Ag/Ab complexes network (C). Such a biosensor can be kept for several months after hydrophilization and several hours (according to antibodies’ lifetimes) after Ab coating in physiological saline without any drift or loss of its properties. General Principles of Immunoelectrode Functioning: Descriptions and Explanations. Antigen/Antibody Binding. Antibodies possess a combining site structure highly complementary to that of their specific antigens. But antibody/antigen interfaces as a whole exhibit poorer shape complementarity when compared to other systems involving protein/protein interactions.11,12 In Ag/Ab binding, antigen specificity depends mainly on the charge distribution carried by the antigenic determinant. The matching of the charged residues and the electrostatic
complementarity also play an important role in the specificity of Ag/Ab complex formation.13,14 But it is clear that charge complementarity is not the only determinant of the binding specificity. Baker and Hubbard15 have suggested that other factors, such as hydrogen bond arrangements, also have a significant role in binding. Braden and Poljak16 confirmed that antibodies bind protein antigens over large sterically and electrostatically complementary surfaces. Van der Waals forces, hydrogen bonds, and occasionally ion pairs provide stability to antibody-antigen complexes. In addition, water molecules contribute hydrogen bonds linking antigen and antibody and increase the complementarity of antigen-antibody interfaces. In addition, Nikolelis et al.17 have reported that Ag/Ab complex formation produces charge neutralization on the antibody molecule. This charge neutralization yields a charge outflow whose intensity and nature depend on those molecules involved in the binding, according to antibody specificity. Davies et al.13 have also suggested that residual water in the molecular structure can also play a significant role in charge dissipation. These charge displacements generate very low current intensities, which can be detected with a low-noise electrode and a highly sensitive current amplifier. Detection of the Ag/Ab Binding by the Immunoelectrode. Any modification in the charge distribution on the antibody-coated electrode surface provides direct evidence of antigen presence. Recognition of antigen by immunoelectrodes presumably involves several physical and chemical processes leading to Ag/Ab complex formation (for a review on immunodetection, see Aizawa,18 Albery,19 Hage20). Antibodies are coated on glassy carbon fibers, the most often used support, or on a Ni/P plate. The electron transference which occurrs on the carbon surface21 and its relationship with atomic structure22 or on the NI/P plate surface suggested that a part of the detected current actually resulted from a surface charge displacement induced by direct and/or indirect ionic movements. Since Ag insertion into Ab molecule caused ions to flow away from the molecule, this flow increased the charge flow expected for Ag/Ab complex formation. This process is, therefore, secondary to the antigen binding but is dependent on the density of antibodies coated on the support. It is expected that each Ab molecule always retains the same number of ion species that will expelled during Ag binding on the Ab. It is also dependent on the properties of the electrode surface itself, i.e., ions available in the liquid medium, ions imprisonned in the carbon or Ni/P network, or ions distributed along the electrode surface. Although the reaction detected could be secondary to the Ag/Ab binding formation, the charge (11) Putnis, A. Introduction to mineral sciences; Cambridge University Press: Cambridge, UK, 1992; 457 pp. (12) Lawrence, M. C.; Colman, P. M. J. Mol. Biol. 1993, 234, 946-50. (13) Davies, R. D.; Sheriff, S.; Padlan, E. A. J. Biol. Chem. 1988, 10, 541-44. (14) McCoy, A. J.; Chandana Epa, V.; Colman, P. M. J. Mol. Biol. 1997, 268, 570-84. (15) Baker, E. N.; Hubbard, R. E. Prog. Biophys. Mol. Biol. 1984, 44, 97-179. (16) Braden, B. C.; Poljak, R. J. FASEB J. 1995, 9, 9-16. (17) Nikolelis, D. P.; Tzanelis, M. G.; Krull, U. J. Anal. Chim. Acta 1993, 282, 527-34. (18) Aizawa, M. Philos. Trans. R. Soc. London 1987, 316, 121-34. (19) Albery, W. Ciba Found. Symp. 1991, 158, 55-67. (20) Hage, D. S. Anal. Chem. 1995, 67, 455R-62R. (21) Hopper, P.; Kuhr, W. G. Anal. Chem. 1994, 66, 1996-2004. (22) Davies, D. R.; Padlan, E. A.; Sheriff, S. Annu. Rev. Biochem. 1990, 59, 45973.
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displacement which occurred during the complex formation is presumably one of the first events related to Ab/Ag complex formation and also the rate-limiting step of those currents detected by the electrode. Simplified Theoretical Approach to the Electrode Response. According to our experiments, current variations are caused by charge displacements directly or indirectly related to antigen binding onto antibody. Thus, the simplified theoretical approach proposed here is based on an estimation of the electrical charges displacements resulting both from specific binding of Ag onto Ab and from nonspecific ion displacements in the surrounding environment during the antigen-antibody complex formation and as well on our previously reported measurements. In the neighborhood of antibodies coated onto the immunoelectrode, antigen molecules were moving randomly, and they likely could collide with an antibody. These antigen molecules are distributed inside a cylindrical layer surrounding the cylindrical electrode (calculations are made for carbon fiber immunoelectrodes). It is assumed that the depth of such a layer depended on the diffusion coefficient of the antigen (D ) 1.66 × 10-6 cm2 s-1 23), which allowed the covered distance per time unit to be calculate (x2 ) 2Dt, with t ) 1 s). The volume, V, containing antigen molecules was defined as V ) (πr22l) - (πr12l). Taking a carbon fiber radius, r1 ) 2.5 × 10-6 m, the electrode radius added to the cylindrical layer radius, r2 ) 20.72 × 10-6 m, and the coated length of the electrode, l ) 7.5 × 10-4 m, V ) 9.97 × 10-10 l. According to the antigen concentration (C ) 5.66 × 10-10 M), there were n molecules (n ) CNAV, with NA the Avogadro number) in the volume V, or 3.4 × 105 molecules in the electrode proximity. The number of possible contacts between the somatostatin molecule24 (antigen) and the antibody per Ag/Ab complex was then estimated. Davies et al.22 had shown that the number of interactions (hydrogen bond and salt link) between charged moieties implied in an antibody/protein complex varies between 2 and 26 (H-bond 2-23; salt link 0-3). To estimate the number of possible H-bonds in the somatostatin molecule, we have made a three-dimensional molecular model. From this model, the hydrogen bond receptor and the hydrogen bond donor which could form a strong bond at the molecule surface could be estimated. The maximum number of contacts per molecule in our molecular complex was 27. Assuming that each contact can induce the release of an equivalent electron, the theoretical electrical charge was calculated from Qc ) neF2725 (with the electron charge e ) 1.6 × 10-19 C, the Faraday’s constant F ) 96500 C mol-1, and the number of contacts per molecule 27, Qc ) 141 nC). The electrical charge measured with the immunoelectrode was Qm ) 126.9 ( 10.34 nC. It should be noted that the calculated value is close to the measured values, even though our hypothesis was optimal, because it is not obvious that all theoretical contacts could be achieved. Moreover, as the number of Ag/Ac complexes increased at the electrode surface, the probability for new contacts decreased since Abs were fixed and cannot be renewed. In our hypothesis, the number of contacts was overestimated, because (23) McDermott, M.; McDermott, C. A.; McCreery, R. L. Anal. Chem. 1993, 65, 937-44. (24) Holladay, L. A.; Puett, D. Proc. Natl Acad. Sci. U.S.A. 1976, 73, 1199-202. (25) Tonnelat, J. Biophysique; Masson: Paris, 1973; p 366.
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Davies et al.22 had found the same actual H-bond number between the antibody and an antigen molecule which was 30-fold larger than the somatostatin molecule. Our proposed current calculation is only a simplified approach because it takes no account of some factors. (1) The antigen binding on the immunoelectrode creates a decreasing gradient of antigen concentration near the electrode which changes the antigen diffusion. (2) The Ag/Ab binding is irreversible in our conditions, and even with an excess of available antigen in the solution, the increasing Ag/Ab complex formation on the surface of the electrode lowers the binding probability of other molecules. (3) The water shell surrounding each involved molecules is a very important property since it may favor linkage. In addition, at very high antigen concentration, the finite antibody number evolves more or less rapidly to an increasing number of saturated antibodies which depends on antigen diffusion and motion (depending on temperature), on the antibody affinity, and on the water shell surrounding antigen molecules. Mariuzza and Poljak26 have demonstrated that the water molecules formed an extended network bridging antigen and antibody and are essential in achieving shape and chemical complementarity between their interacting surfaces. Goldbaum et al.27 in their model showed that water molecules bound to the antigen and the antibody are conserved upon complex formation and provide bonds which are important for the stability of the complex. This is in agreement with a model in which binding of antigen to antibody is accompanied by the release of a very small number of water molecules. These water molecules evacuated during Ab/Ag complex formation could alter the electrical charge distribution on the electrode surface. Water molecules are the solvent of a physiological ionic solution, so water motion induces electrical charge motion. In their work on the three-dimensional structures of the Fv fragment of the anti-hen egg white lysozyme antibody in its free and antigen-bound forms, Bhat et al.28 have reported that these structures reveal a role for solvent molecules in stabilizing the complex and provide a molecular basis for understanding the thermodynamic forces which drive the association reaction. Four water molecules are buried, and others form a hydrogen-bonded network around the interface, bridging antigen and antibody. The large number of solvent-mediated hydrogen bonds, in conjunction with direct protein-protein interactions, should generate a significant enthalpic component. Thus, the main forces stabilizing the complex arise from antigen-antibody hydrogen bonding, van der Waals interactions, enthalpy of hydration, and conformational stabilization rather than solvent entropy (hydrophobic) effects. Braden et al.29 have also shown, through a study of the conservation of water molecules in crystal structures of an antibody fragment, free antigen (lysozyme), and complexed antigen, that among 99 water molecules of the antibody-antigen complexes, the antibody-lysozyme interface includes 25 well-ordered solvent molecules, which are bound directly or through other water molecules to both antibody and antigen. The 25 well-ordered (26) Mariuzza, R. A.; Poljak, R. J. Curr. Opin. Immunol. 1993, 5, 50-5. (27) Goldbaum, F. A.; Schwarz, F. P.; Eisenstein, E.; Cauerhff, A.; Mariuzza, R. A.; Poljak, R. J. J. Mol. Recognit. 1996, 9, 6-12. (28) Bhat, T. N.; Bentley, G. A.; Boulot, G.; Greene, M. I.; Tello, D.; Dall’Acqua, W.; Souchon, H.; Schwarz, F. P.; Mariuzza, R. A.; Poljak, R. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1089-93. (29) Braden, B. C.; Fields, B. A.; Poljak, R. J. J. Mol. Recognit. 1995, 8, 317-25.
interface water molecules contribute a net gain of 10 hydrogen bonds to complex stability. In addition to contributing hydrogen bonds to the antibody-antigen interaction, the solvent molecules fill many interface cavities. Braden et al. also noticed that 15 water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and five water molecules are added at the antibody-antigen interface with the formation of the complex. Then, Ab/Ag complex formation induces building of H-bonds not related to antigen or antibody structure, and numerous hydrogen bonds between antigen and antibody residues are mediated by water molecules, thereby neutralizing practically all unpaired atoms in the interface.26 The motion of water molecules during Ab/Ag binding displaces charged solvated molecules (ions) which also induce electrical charge displacement. But another electrical charge could be due to rearrangements in solvent or protein structure or to secondary interactions involving other residues, as suggested by Goldman et al.30 to explain the two kinds of coupling energy measured in hydrogen bonds implied in interactions occurring in an idiotope-antiidiotope proteinprotein complex. The relatively small difference between calculated and measured electrical charges might be explained by the wide variety of molecular interactions described above, but it does not affect our conclusion, because for one given antibody and one given antigen, the water molecules network and the number of molecules shifted are established and constant. Treatment of the Electrode Response. The carbon fiber electrode responses appeared as transient peaks on plots. Transient peaks (Figure 4 A-C) were directly related to antigen concentration in somatostatin-14 ranging from 10-15 to 10-13 M. For an antigen concentration ranging from 10-12 to 10-6 M, the peak amplitude did not increase, but the transient current was of longer duration. This observation suggested that the electrode response reflected the quantity of peptide available close to the electrode according to the peptide diffusion coefficient. The quantity of available peptide depended on the antigen concentration gradient created by the diffusion and the Ab/Ag complex formation on the electrode, the quantity of which is timedependent. The transient currents were therefore time integrated, and the results are expressed in nanocoulomb. The calibration curves are then more smooth, and variations steps due to fast changes of the concentration are more reliable, because they represent the total amount of electrical charges removed from the antibody and electrode surface. Dose-Response Curve and Controls. Before doseresponse curve establishment, we checked immunoelectrode specificity. Indeed, our purpose was primarily to detect somatostatin among many kinds of proteins present in the brain fluid, so it was necessary to ensure that the immunoelectrode response was not influenced by some unwanted proteins. Some control experiments were performed (1) with somatostatin applied to either noncoated electrodes or to electrodes coated with antibodies raised against insulin and R-fetoprotein, and (2) with insulin and R-fetoprotein added to anti-somatostatin-coated electrodes. No response was ever observed under these conditions. The same kind of control experiments were performed with anti-histamine immunoelectrodes (Figure 7D,E). (30) Goldman, E. R.; Dall’Acqua, W.; Braden, B. C.; Mariuzza, R. A. Biochemistry 1997, 36, 49-56.
Figure 4. Calibration curve of the anti-somatostatin immunoelectrode and examples of electrode signal in response to 60 µL of somatostatin addition [(A) 10-15, (B) 10-12, and (C) 10-9 M, indicated by vertical arrows] to 1 mL of snail Ringer saline. The horizontal dashed line is the mean baseline current integrated over a 60-s period prior to peptide addition. The shaded area is the electrical charge of the response. Horizontal axis is log of the amount in picomoles. (D) Dose-response curve from six experiments similar to those shown in A, B, and C.
Figure 5. Electrode response to different physiological pH values (6.8, 7.3, 7.5, 7.8, 8.0) of 20 electrodes. Somatostatin-14 concentrations tested are in a range from 10-13 to 10-8 M. Horizontal axis is log of the amount in picomoles.
Dose-response curves were carried out by adding 60 µL of somatostatin-14 (Sigma) at concentrations ranging from 10-15 to 10-7 M in 1 mL (final concentration 6 × 10-17-6 × 10-9 M) of snail Ringer saline. After each measurement, 60 µL of the solution was withdrawn so as to keep a constant volume. The binding of peptide to the immunoelectrode generated a transient current of a few nanoamperes (Figure 4). The transient currents lasted as long as free peptide molecules were still present in the environment close to the electrode and as far as a steady equilibrium was established between the linked antigen and free antigen according to the antigen diffusion coefficient. This means that Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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Figure 6. Examples of detection in fluid media. Typical response of anti-histamine immunoelectrode in different histamine concentrations in PBS [(A) 10-10, (B) 10-9, and (C) 10-7 M, indicated by vertical arrows]. Comparison between recording with (A, B) or without BSA (C) and different controls [(D) PBS only, (E) carnosine].
the electrode response is related to the amount of peptides, that is, to the antigen concentration in the bulk. The transient currents were, therefore, time integrated, and the results were expressed in nanocoulombs. The dose-response curve was obtained from six similar experiments in which various amounts of somatostatin14 were randomly added. Immunoelectrode Response to pH Changes. Immunoelectrodes were tested in PBS at various pH solutions. In these tests, we have used the mammalian physiological range of pH values: 6.8, 7.3, 7.5, 7.8, and 8.0. Twenty similar anti-somatostatin immunoelectrodes were tested in 1 mL of each solutions. For each pH value, somatostatin-14 concentration varied in a range of 10-13-10-8 M, with continuous stirring. In this physiological range of pH values, except at 6.8, all immunoelectrodes gave the same kind of responses (Figure 5) in a range of 0-230 nC. At pH 6.8, the electrical charge is less than 200 nC, but the curve presented the same features but with a smaller slope. Therefore, in the mammalian physiological range, pH variations did not induce any shift in the immunoelectrode response, and 6.8 is a bit too acidic for an optimal Ab/Ag complex formation. Application of Ni/P Plate Electrode for Ag Detection in Fluid Media. We have worked in two steps with either fiber electrodes or plate electrodes. We have first compared the performances of carbon fiber and Ni/P plate immunoelectrodes. Plots of current changes caused by different somatostatin-14 concentrations were obtained for both types of immunoelectrodes, and they appeared quite similar one to another. There is no difference in protein detection sensitivity between the two kinds (31) Malmsten, M.; Lindman, B.; Holmberg, K.; Brink, C. Langmuir 1991, 7, 2412-4.
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of electrode. Also both kinds of electrodes can be equally used in Ag detection. Second, we have compared measurements of histamine concentration in two different physiological media, one PBS (pH 7.4) and the other PBS containing 80 g/L of bovine serum albumin (BSA), which is the normal blood protein concentration, and BSA was used as a reduced model of serum. Current variations were very similar in all the cases (Figure 6). BSA could be linked to the electrode by a nonspecific linkage. Nevertheless, these facts did not compromise interpretation of results. Indeed, Malmstem et al.31 had tested BSA as blocking agent against nonspecific adsorption after antibody coating on a polystyrene surface, and they showed that BSA did not disturb antibody binding. Advantages and Use of the Two Types of Immunoelectrode. Glassy carbon fiber microimmunoelectrodes, glued on a copper wire, have been conceived for in situ detection. The smallsized carbon fibers (5 µm) allow a cellular approach without visible damage. The advantage of single-fiber electrodes is their very small size; consequently, such an electrode can be readily handled in close cellular environment. But it is more breakable than multifiber electrodes. An increase in the number of fibers raises the surface area available to antibodies coating. The carbon fiber, though it is an excellent adsorbant material suitable for antibody coating, suffers several disadvantages: its conductivity is lower than that of metal, it is not easy to deposit carbon on a solid metallic support of small size, and it is thus a breakable material inadequate for analysis in media other than fluids. CONCLUSION Immunodetection by use of immunoelectrodes is a promising technique since it allows real-time detection of low protein amounts, especially neuropeptides or neurotransmitters which as yet needed time-consuming and/or destructive methods. Considering the remaining problems of antibodies quality and specificity and the homogeneity improvement of a solid adsorbant surface, we have obtained satisfying results in term of sensitivity and reliability. Improvement should be obtained from better knowledge (1) of antibodies properties during their linkage to a surface different from a cell membrane and, conversely, (2) about the cells’ reaction toward a large amount of antibodies concentrated in a very small volume in the cell environment. This technique allows detection of low antigen amounts (femtomoles). But we can easily conceive antibody detection using binding of antiidiotypical antibody to an immunoelectrode. Binding improvement may be obtained by methods analogous to classical immunoamplification, i.e., multiple antibodies binding to biotin-avidin complex. Developments of new, very sensitive methods of direct protein detection inside tissues such as muscle or blood are expected by many scientists and can result from immunoprobes improvement. Received for review May 5, 1998. Accepted September 11, 1998. AC980492M
