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Anal. Chem. 1882, 64, 997-1003
Real-Time Monitoring of Immunochemical Interactions with a Tantalum Capacitance Flow-Through Cell Andreas Gebbert,* Manuel Alvarez-Icaza, Walter Stocklein, and Rolf D. Schmid Gesellschaft fiir Biotechnologische Forschung (GBF), Department of Enzyme Technology, Mascheroder Weg 1, 3300 Braunschweig, Germany
A flow-through cell for the real-tlme capacltance monltorlng of lmmunochemlcal lnteractlons has been developed. It consists of a tantalum strlp onto which tantalum oxlde was grown electrochemlcally to a layer thlckness as small as 5 nm. Antlbody or antigen was hnmoblllzed onto the tantalum oxlde surface, and blndlng of the correspondlng analyte resulted in modlflcatlon of the ekctrlcal capacltance of the system. Wlth mouse IgG as the Ilgand, real-tlme monltorlng of antl-mouse-IgG In the nanogram per mllllllter range was poedble. The behavlor of the system wlth respect to analyte dze and concentratlon was Investigated. The tantalumAantalum oxlde/electrolyte system can be manufactured easlly and reproduclbly.
In the article presented here, the dielectric is grown directly on the surface of a tantalum substrate by electrochemical oxidation. This technique allowed the controlled production of very thin (as low as 5 nm)tantalum oxide layers which form the dielectric surface of highly sensitive capacitive sensors. THEORETICAL BACKGROUND The electric capacitance between two parallel plates separated a distance d is given by the equation
C = c,eA/d
(1)
where e, is the permittivity of free space, e is the dielectric constant of the material between the plates, and A is the surface area. Large capacitances may be achieved by reducing the distance between the two electrodes. The formation of constantly and reproducibly short (micrometer range) distances between INTRODUCTION the two plates is mechanically limited. The limitation does not apply to electrolytic capacitors, where one of the plates The important role of i"unoassays1s2 especially in clinical is covered with an insulator and the second plate, or counter and pharmaceutical,3environmental,4and bioprocess5analysis electrode, is represented by an electrolyte (Figure 1). The is based on their high sensitivity and specificity, allowing capacitance is then determined by the thickness and dielectric simple and rapid detection of a broad spectrum of analytes properties of the insulating layer and the solid/solution ineven in a complex sample matrix? A large variety of assay terface, both of which constitute the dielectric. Hence, an formats has been developed, which include applications of electrolytic capacitor allows the detection of material bound radioimmunoassay (RIA),7 enzyme immunoassay (EIA)? to a plate; this opens the possibility of detection of any analyte fluorescence immunoassay (FIA)? and the enzyme-modified binding to specific ligands immobilized on the insulating layer immunoassay techniques (EMIT),1°as reviewed by Gosling of that plate. As an example, the binding of antigen to imet aL3 mobilized antibodies is shown in Figure 2a. In such a sensor, Several attempts have been made to measure the antithe space between the immobilized protein molecules is filled body-antigen or antibody-hapten binding directly without with electrolyte; i.e. there is a conductor connected in parallel the use of labeled compounds. Optical,l'-'' electrochemical,1s with the capacitor, as illustrated by the model represented and micromechani~al~*~~ methods have been investigated. in Figure 2b. Therefore, it is necessary to use a measuring These methods allow in principle real-time measurements of method or instrument that can distinguish between changes ligand-analyte binding. Fundamental chemical and physical limitations of direct assays are described by E d d o ~ e s , ~ ~ in capacitance and changes in conductance due to their different phase characteristic^.^^ K ~ o y m a nand , ~ ~B e r g ~ e l d . ~ ~ The sensitivity of the sensor increases with decreasing The concept of capacitive affiiity sensors, with which this thickness of the insulating layer. This can be demonstrated paper deals, has been recently It is based on by considering the initial capacitance, Ci, formed by the dithe principle that for electrolytic capacitors the capacitance electric and the antibodies fiied to the dielectric surface, on depends on the thickness and dielectric behavior of a dielectric one hand, and the capacitance, C,, due to the binding of the layer on the surface of a metal plate. As will be explained in antigen, on the other. The total capacitance is given by the the Theoretical Background, the capacitance and the sensirelation for two capacitors connected in series: tivity of the device are inversely related to the thickness of the dielectric. In previous the dielectric layer attached to the conductor was produced using technology from the microelectronics industry and the material to which or the dielectric was attached was a semiconductor instead of a conductor. The reported sensors require a polarization voltage for their operation. Another disadvantage is that the (3) infrastructure for the production of the sensor chips is not accessible for most of the laboratories working in the develwhere Ct is the total capacitance,measwed through the sensor opment of biosensors. Looking for an alternative device, terminals. The sensitivity is given by the ratio of change of Newman et al.% used a copper electrode covered with parylene the total capacitance Ct to the change in capacitance due to and silicon monoxide layers as insulator. In this case, the the antigen binding. For that purpose the partial derivative thickness of the dielectric (1.3 p m ) as well as the reproducof Ct with respect to C, should be considered: ibility of its manufacture is assumed to be mechanically limited. 0003-2700/92/0364-0997$03.00/00 1992 American Chemical Society
998
ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
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Figure 1. Concept of an electrolytic capacitor: (a) schematic and (b) electric description.
elution buffer
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u
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Pump Flgure 3. Flow immunocapacitance system.
For a molecule with a radius of 100 A, the approximate value for an antigen-antibody complex, estimated from IgG dimensions of 142 A X 85 A,40the relaxation time calculated using eq 5 is 3.1 ps. In light of the fact that the relaxation time is related to frequency by the equation
caQ Ci -.
7
7-
Figure 2. Representation of the analyte binding to antibodies immobilized onto the sensor surface (a) and the electric model used to represent it (b).
The sensitivity tends to 1when Ci is very large compared to C,, this means that a change due to the binding of antigen will produce a maximum change in the sensor output. Therefore, the initial capacitance which is formed by the insulating layer and the antibody layers has to be very large. The capacitance of the antibodies depends on properties that cannot be used as design parameters. The only parameter left to be optimized from a design point of view is the thickness of the insulating layer, which in order to have a maximum capacitance should have a minimum thickness and be nonporous and nonsoluble. Generally, the formation of an insulating oxide layer on a metal substrate can be classified into two groups:37 the barrier-type, which gives a thin, nonporous layer of oxide, and the porous-type, which brings about a thick, porous oxide layer. The first type of oxide layer is produced by the formation of tantalum oxide on tantalum substrate.38 Therefore tantalum might be a useful material to construct a highly sensitive capacitance sensor. Finally, with regard to the design of the sensor, it is necessary to consider the behavior of proteins as dielectrics in the presence of electric fields. The dielectric constant of proteins, around 20,39should be large enough to obtain an observable capacitance change with the sensor when the analyte is a protein. On the other hand the capacitance measurement of proteins might be limited by the time required for the protein to move and polarize under the influence of the electric field. A simple physical model, described by Pethig and Kell,39can be used to gain a mental picture of the processes occurring during the movement of molecules with a permanent dipole moment in response to electric fields. Approximating the shape of the molecule using a rigid sphere of radius a, turning in a Newtonian hydrodynamic fluid of macroscopic viscosity 8,and using the Stokes-Einstein relation for the molecular friction coefficient, these authors found that the relaxation time T for a molecule is given by the equation r
= 4?rOa3/kT
(5)
where k is the Boltzmann constant and T the absolute temperature. At 293 K the viscosity of water is kg m-l s-l.
= 1/2?rV0
(6)
a frequency of 51 kHz would be the maximum frequency at which free antigen-antibody complexes respond to changes in the electric field. However, when the antibody is immobilized, the antigen-antibody complexes do not rotate but they will have to pivot around the point where the antibody is immobilized. The pivoting radius is, therefore, the whole length of the complex (200 A). For this rotation radius the maximum excitation frequency can be calculated to be around 6 kHz. The restricted flexibility of immobilized molecules has to be considered, so that an even lower frequency might be expected. On the other hand, capacitance measurements are difficult for frequencies below 20 Hz. Therefore, the optimal frequency to observe changes in capacitance due to the binding of antigen to an antibody should be in the region between 0.02 and 6 kHz and has to be determined experimentally. EXPERIMENTAL SECTION Reagents. The following immunoreagents were used: mouse IgG and goat-anti-mouse IgG (GAM) from Sigma; sheep-antimouse IgG-@-galactosidase(SAM-GAD) from Amersham. Albumin fraction V was obtained from Boehringer, cyclohexyl(2morpholinoethy1)carbodiimidemetho-4-toluenesulfonate(research grade) from Serva. All other chemical reagents were of analytical-gradepurity and were obtained from either Merck or Aldrich. The standard buffer was 30 mM sodium phosphate, 100 mM NaC1, pH 7.5. Tantalum foil (0.1 mm X 100 mm X 200 mm; 99.9+%) was obtained from Heraeus. ' Analytical System. The analytical system (Figure 3), in which the capacitance flow-through cell was integrated, consisted of the following apparatus: two 3/2 way magnetic valves from Lee Co. (Westbrook, CT), a minipuls peristaltic pump (Gilson),a Hewlett Packard impedance meter HP 4284A, and an IEEE 488 interface which connected the measurement system to a computer, where values of capacitance and conductance of the flow-trough cell were stored. Design of the Flow-Through Capacitance Cell. A schematic diagram of the flow-throughcell is shown in Figure 4. Two sensor strips (1cm X 4 cm), consisting of the tantalum substrate ' (1)onto which tantalum oxide (2) was grown, were separated by a Teflon spacer (d = 1mm) (3). The spacer was designed with a special shape (top view Figure 4b) to prevent entrapment of air bubbles in the flow-through cell. The sample fluid flows through the spacer from the broader to the narrower end. During passage through the cell chamber, the flow velocity increases, producing a pressure difference between the top and the bottom of the air bubble. Therefore, and due to the vertical orientation of the cell, the bubble will be forced to the exit of the chamber. The sensor parts were clamped together with two Teflon plates
ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
bottom
00.0'
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Figure 4. Flow-through capacitance cell: (a) vertical section; (b) horizontal section; (1) tantalum foil; (2) tantalum oxide; (3) Teflon spacer; (4) Teflon plates; (5) metal box. light interference
1300 1200 1100
(7)
where d, is the thickness of the oxide layer, XI and X2 the wave lengths of two consecutive peaks, n the refraction index of tantalum oxide (2.5),and a the angle of the emitted light. Figure 7 shows a typical result obtained by the formation of tantalum oxide on tantalum substrate for a fixed current density of 2 mA cm-2. During the formation the capacitance decreased due to the growth of the oxide layer. The thickness d of the oxide layer, examined by analyzing optical interferencespectra, was increasing with time and can be calculated from the formation voltage using the following equati~n:~~ (8)
where a and b are constants determined experimentally, by a
900 800 700
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(4). The tantalum electrodes were connected via the four-electrode te~hnique.~~ Finally, the sensor was shielded by a metal box (5). Preparation of the Sensor Strip. (a) Manufacture of the Tantalum Oxide Layer. Tantalum strips were cut and cleaned for 30 s in 48% hydrofluoric acid. The anodic formation of tantalum oxide on the tantalum substrate was made in 4% boronic acid stirred in a beaker using a platinum counter electrode. Different thicknesses of oxide layer on the surface were obtained by varying the formation voltage from 0.1 to 500 V using a formation time of 1 h, unless otherwise indicated in the text. (b) Characterization of the Oxide Layer Surface. (1) Interference Spectra. In contrast to aluminum oxide, which represents the cheapest and most common dielectric for commercial electrolytic capacitors, the solubility of tantalum oxide in the electrolyte is negligible and a highly uniform oxide layer is formed, hence creating very thin and homogeneous layers on a surface which results in optical light interferences (Figure 5). The color of the sensor surface changes over a wide spectrum depending on the formation time. The thickness of the layer can be determined by analyzing the optical interference spectrum of the surface. The spectra of the generated surfaces were analyzed with a Shimadzu spectrophotometer RF 5000. A typical interference spectrum obtained by the formation of tantalum oxide on tantalum is shown in Figure 6. The thicknesses of oxide layers were calculated by the following equation:42
1000
450
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Figure 5. Schematic representation of the optical light interference caused by a homogeneous, nonporous layer of tantalum oxide on a tantalum substrate.
d=a+bV
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formation time of 1 h, a = 5 f 3 nm and b = 1.99 f 0.1 nm V-l. It can be predicted from Figure 7 that a high sensitivity to changes in thickness of the dielectriclayer, measured by changes in capacitance,can be obtained for formation voltages below 250 V. (2) Scanning Electron Microscopy (SEM). Scanning electron micrographs of the tantalum oxide surfaces produced were taken using a Zeiss DSM 940 SEM instrument. For comparison, aluminum, as a common substrate for electrolytic capacitors, and tantalum oxide layers were grown under the same formation conditions (Figure 8A,B). The generated tantalum oxide surface, in contrast to the aluminum oxide surface, was uniform and nonporous. On the other hand some irregularity of the surface structure can be observed probably due to mechanical stress. (c) Immobilization of Protein. A covalent attachment of the antibodies to the surface was obtained by silanization of the surface and coupling the antibody to the silanized surface with carbodiimideP For silanization the formed tantalum strips were incubated for 3 h in (3-aminopropy1)triethoxysilane (2% v/v in acetone), rinsed three times with acetone, and dried for 2 h at 65 "C. The strips were then immersed for 2 h in a mixture of 1 mg of antibody and 15 mg of cyclohexyl(2-morpholinoethy1)carbodiimide metho-p-toluenesulfonate in 6 mL of 30 mM phosphate buffer, pH 4.0. Finally, nonspecific binding sites were blocked by incubation of the sensor strips for 1 h in 0.1 M lysine, pH 7.5. The capacitance measurements were not affected by using 1%BSA or 0.1 M glycine, pH 7.5, instead of lysine. Protein was determined in the incubation solution by the bicinchoninic acid (BCA) method (Pierce). The total protein binding to the sensor mol cm-2). surface was 2 pg cm-2 (1.33 X (a) Characterization of the Biological Behavior of the Prepared Surface. After formation of the tantalum surface and immobilization of antibodies, the sensor strips were used for a standard fluorometric immunoassay to check the biological behavior of the sensor surface. The strips were incubated for 10 min in a solution of antigen conjugated to &galactosidase (0.2 pg/mL-l) and washed three times with buffer (0.1M phosphate, 0.15 M NaC1, 0.1% Tween, pH 7.5). Then 4-methylumbelliferyl 6-D-galactopyranoside (0.3 mM) was added as substrate. After
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was applied because it was found to be optimal, as will be shown at the end of the Results and Discussion. Drifts in the base line during the measurements were calculated and mathematically compensated.
Flgure 8. Scanning electron micrographs of metal oxide surfaces, grown in 4% boronic acid using a fixed formation voltage of 300 V for 1 h: (A) aluminum oxide; (B) tantalum oxide. Bars represent 20 pm (magnification factor of enlarged inserts: 4X).
5 min of incubation the product was measured fluorometrically (AEx = 360 nm; XEm = 450 nm). The surface was regenerated by elution of bound analyte with glycine/HCl (0.1 M, pH 2.1) for 3 min. The activity of the sensor strips decreased to 50% after 40 measurements. With control strips containing immobilized BSA, nonspecific binding of the analyte was observed with an average peak height of 15%. Analytical Procedure. The system was first rinsed with standard buffer. The flow rate was 0.3 mL min-' over the measurement duration. After switching to the sample fluid and incubating the sensor surface with the analyte for 10 min, which corresponds to a sample volume of 3 mL, the flow was returned to the standard buffer (pH 7.5). After the measurement, elution of bound antigen with elution buffer (0.1 M glycine/HCl, pH 2.1) for 5 min regenerated the sensor for the next measurement. Changes in capacitance of the tantalum flow-through cell were observed continuously with the impedance meter using an excitation voltage of 100 mV. This low potential was selected to avoid further growth of the oxide layer. A frequency of 1 kHz
RESULTS AND DISCUSSION ( 1 ) Detection of Sheep-Anti-Mouse IgG-8Galactosidase (SAM-GAD). The possibility of detecting the binding of an analyte to the sensor strip was tested with sheep-anti-mouse IgG-P-galactosidasedue to both its relatively large size (ca.MW 450 OOO) and its usefulness for fluorometric reference measurements. Tantalum strips were formed for 1 h with a 250 V formation voltage (which corresponds to a thickness of tantalum oxide of approximately 500 nm), and mouse IgG was immobilized on the surface of the sensor as described in the Experimental Section. A solution of 20 ng mL-l sheep-anti-mouse IgG-@galactosidase in standard buffer was passed through the cell, and the capacity change was measured continuously (Figure 9). The binding of analyte produced a thicker dielectric layer on the interface of the surface; the capacitance decreased as new material was attached to the surface. Two mechanisms can be proposed for the attachment of material, both specific as well as nonspecific binding of protein. For the interpretation of the capacitance curve obtained with the described system, we have to consider that both the immobilized protein and the analyte were heterogeneous (different mouse IgG subclasses, polyclonal antibodies). Therefore, different affinity constants and dissociation rate constants can be assumed for the different fractions of antibody and antigen. Rinsing with standard buffer produced an increase in capacitance. This increase may be due to loosely bound protein, i.e. nonspecific bound protein or fractions of specific bound protein with a high dissociation rate constant. After a few minutes an apparent equilibrium was reached. The difference between the final and initial capacitance gives information about the amount of strongly specifically bound analyte. (2) Investigation of Nonspecific Binding. Tantalum strips were formed at 10 V, corresponding to a layer thickness of 25 nm. To investigate nonspecific binding phenomena, specific as well as control sensor strips were compared, on which mouse IgG and BSA were immobilized respectively. A third strip was used without any protein layer. The three strips were used for the measurement of 20 ng mL-l sheepanti-mouse IgG-@-galactosidase(Figure 10). The sensor surface without immobilized protein gave no capacitance change due to the incubation with antigen, while the surface with immobilized BSA produced a capacitance change of 15%, as compared to the IgG sensor, due to nonspecific binding of
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protein. This value is in good agreement with the fluorometric assay obtained by the control fluorometric experiments described in the Experimental Section. Another approach to examine to which extent the binding of SAM-GAD (20ng d - l ) to the IgG sensor strip was specific and to exclude interference on the signal due to the buffer was to determine the binding of analyte which had been denatured by boiling for 15 min in a 100 O C water bath (Figure 11). During the f i t few minutes of incubation the denatured protein caused a capacitance change similar to that obtained with the nondenatured analyte. In contrast to the nondenatured protein the capacitance change reversed largely after switching to the standard buffer. The binding of the denatured protein estimated by the difference of initial and final capacitance was in the range 10-20% as compared to the case of the nondenatured protein. (3) Analyte Concentration Dependence of the Capacitance Change. To observe the relationship between capacitance changes and concentration of analyte, different concentrations of sheep-anti-mouseIgG-@-galactosidasewere prepared. Mouse IgG sensor strips were prepared as described (see section 1 above). The analyte solutions were measured starting with the lowest concentration. After each measurement, the sensor was regenerated with glycine/HCl, 0.1 M, pH 2.1 (Figure 12a). The obtained capacitance curves indicated two effects. The difference between the initial and final capacitance increased with analyte concentration. The slope of the decrease of the capacitance during the analyte
Figure 12. Detectlon of various analyte concentratlons: (a, top) reai-time capacitance curves; (b, bottom) dependence of capacitance change on analyte concentration.
incubation also increases with higher concentrations of analyte, which implies that the binding of analyte to the sensor surface is faster at higher analyte concentrations. Therefore, determination of analyte concentration via this slope might be a procedure to decrease the time required for the assay. Figure 12b shows the relationship between the difference of the initial and final capacitance and the concentration of analyte. The measuring range was about 0.2-20 ng mL-'. A similar curve (not included here) was obtained by plotting the slope of the curves (5-25 pF min-') during the analyte incubation against the concentration of analyte. In order to avoid matrix effects during the incubation of the sample, the comparison of the initial and f i i capacitance in the same buffer (pH 7.5)ii preferable. However, the limit of detection is still defined by the nonspecific interactions. On the other hand a high efficient washing step with buffer should elute nonspecifically bound substances, which could influence the measurement. Methods for improving the efficiency of this washing step (e.g. detergents, different flow rates, ultrasound) are under investigation. (4) Measurements Using Highly Sensitive Sensor Strips. To show experimentally how the sensitivity of the sensor can be increased by manufacturing thinner layers of tantalum oxide, a dielectric was grown over night with 100 mV to a layer thickness of about 5 nm. Mouse IgG was immobilized onto its surface, and a dilution of 20 ng mL-' goat-anti-mouse IgG in standard buffer was measured (Figure 13). The figure shows the relatively high capacitance change even for the detection of the nonlabeled antibody (MW 15OOOO), indicating that sensitivity of the sensor was improved by the reduction of the oxide layer thickness, as described above. On the other hand, it must be considered that also
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the sensitivity to parameters which interfere with the measurement increased, e.g. changes in salt concentration during the assay. Furthermore, the mechanical stability of the oxide layer decreases with decreasing thickness. From this points of view a thickness of less than 5 nm is not practicable. Figure 13 also shows that a doubling of incubation time caused a doubling in the difference of the f i i and initial capacitance. Thus, increasing the incubation time may be a further way to increase the sensitivity of the sensor for the detection of low levels of analyte. (5) Optimization of the Excitation Frequency. Information about the optimal excitation frequency was gathered by the following experiment. Mouse IgG was immobilized on a 250-V-formed tantalum strip. Frequencies from 50 to 20 OOO Hz were applied for the detection of sheep-anti-mouse IgG&galactosidase (Figure 14). For this analyte an excitation frequency of lo00 Hz resulted in a maximum change in capacitance; hence this frequency was used for experiments. CONCLUSION We have shown the feasibility of using the tantalum capacitance sensor for the detection of proteins by immunochemical binding reactions. The system should also be useful for the investigation of other ligand-analyte systems (lectins-carbohydrates, receptors-peptides) or DNA-DNA hybridization studies. The data obtained can be used to indicate the presence of an analyte and to calculate ita concentration. The results suggest the possibility that in the future capacitance measurements might be used to characterize the binding reaction via estimation of affinity constants and dissociation rate
constants from the slopes of the associated capacitance changes. There are, however, some pointa which have to be improved. The system, as it stands, is limited to detect relatively large molecules (MW 15OOOO or larger) even with a sensor sensitivity based on a dielectric thickness near the theoretical limit. This limit should be around 3 nmSu For such a distance, electrons can be transferred, by tunneling through the dielectric barrier, at such a rate that the dielectric cannot be considered as such any more. In addition, the precision of capacitance measurements that can be obtained with commercial instruments (one part in 1 0') is better than the level of noise that was detected. Thus, the limit of detectability depends more on noise control than on a lack of sensitivity. In our system, most of the noise appears to be related to the fluid or ita movement. Turbulence inside the measuring chamber can create, by cavitation, microbubblea to which the capacitance measurement is sensitive. The presence of small solid particles or gas in the fluid can be another source of noise, and other effects like streaming potential have to be considered. A better deaign of the measuring cell hydrodynamics together with filtration and degassing of the liquids may result in a significant improvement of the detectability that can be achieved with this system. From the chemical aspect, immobilization methods on tantalum oxide surfaces can still be improved. Antibodies can be immobilized on silica surfaces by a number of well-developed methods, allowing the surface to be regenerated hundreds of times. These methods have been applied also for metal oxide surfaces like aluminum and tantalum. However, it can be assumed that the effectiveness of binding with respect to antibody load and regenerability of the sensor surface can be improved by designing immobilization methods especially for the respective metal oxide surface. After these improvements have been achieved, the minimum detectable size of analyte molecule could be determined. Finally, this measurement method shares with all other direct immunoassay systems the problem that the signal is sensitive to unspecific binding. In the capacitance approach, however, a solution might be offered on the basis of the different frequencies to which molecules can respond. By analyzing the changes in capacitance a t different excitation frequencies, it might be possible to have an estimation of the spectrum of sizes of material that are deposited on the capacitor surface. With this information and the knowledge of the approximate size of the antigen-antibody complex, it should be possible to calculate the amount of material specifically bound to the surface. Work in this direction is already in progress. ACKNOWLEDGMENT We thank D. Hanisch and H. Schillig for assistance in manufacturing the flow cell and Dr. H. Liinsdorf for carrying out the SEM. We also wish to acknowledge R. Hanke from the Physikalisch-Technische-Bundesmstalt Braunschweig who offered us the use of their capacitance measurement systems during the initial stage of the project and W. Koschinski from the Institut fiir Halbleitertechnik und Optik, TU Braunschweig, for helpful discussions in the interpretation of the interference spectra. This work was fiiancially supported by Braun Diessel Biotech gmbH, 3508 Melsungen, FRG, and the BMFT Germany. Registry No. Tantalum,7440-25-7;tantalum oxide, 59763-75-6. REFERENCES (1) (2) (3) (4)
North, J. Trends Biotechnd. 1885, 7, 180-188. Alzawa, M. Phil. Trans. R . Soc. London, B 1087, 316. 121-134. Goaling, J. P. Clln. Chem. 1900, 8 . 1408-1427. Vanderlaan, M.; Watklns, E.; Stanker, L. Envlron. Scl. Technd. 1988, 22, 247-254.
Anal. Chem. 1992, 64, 1003-1008
(5) Bradley, J.; Stkkleln, W.; Schmld, R. D. Roc. Confr. Qual. 1991, 1 , 157-183. (8) Tljssen, P. Laboratory Techniques in Biochemktry and Molecular Biology; Elsevler: Amsterdam, 1985; Chapter 1. (7) Chard, T. An Infroductfon to RadEolmmunoassey and Rehted Techniques; Elsevler: Amsterdam, 1987. (8) Oellerlch, M. Methods of Enzymatic Analysis, 3th ed.;Verlag Chemle: Welnheim, 1983; Chapter 2.7. (9) Hemmilae, 1. Clln. Chem. 1985, 31, 359-370. (IO) Rubenstein, K. E.; Schnelder, R. S.; Ullman, E. F. Biochem. Res. Commun. 1972. 47, 846-851. (11) Mane, M.; Cleudel, J. P.; Clret, P. Clln. Chem. 1987, 33, 209-213. (12) Collet-Cassart. D.; Limet, J. N.; Van Krleken, L.; De Hertogh, R. Clln. Chem. 1080, 35, 141-143. (13) Kooyman, R. P. H.; Lenferlnk, A. T. M.; Eenlnk, R. G.; Greve, J. Anal. Chem. 1091, 63. 83-85. (14) Lukosr, W. Fresenius’ J. Anal. C h m . 1990, 337, 24-25. (15) Boever, J.; Kohen, F.; Bouve, J.; Leyseele, D.; Vandekerckhove, D. Clln. Chem. 1900, 36, 2036-2041. (16) . . Brbht. F. V.: Betts. T. A.; Lltwller. S. Anal. Chem. 1990, 6 2 , 10&5-1069. (17) Fagerstam, L. I n Technlques In Protein Chemistry II; Vlllafranca, J. J., Ed: Academlc Press: New York, in press. (18) Janata, J.; Blackburn, G. F. Ann. N.Y. Acad. Scl. 1984, 428, 286-292. (19) Borman, S. Anal. Chem. 1987, 59. 1161-1164. (20) Davls, K. A. Anal. Chem. 1989, 61, 1227-1230. (21) Muramatsu, H.; Dlcks, J. M.; Tamiya, E.; Karube, I. Anal. Chem. 1987, 59. 2760-2763. (22) Wohltjen, H.; Dessy, R. Anal. Chem. 1979, 51, 1458-1464. (23) Wohltjen, H.; Dessy, R. Anal. Chem. 1979, 51, 1465-1474. (24) Kurosawa, S.; Tawara, E.; Kamo, N.; Ohta, F.; Hosokawa, T. Chem. hem. Bun. 1990, 38, i i i 7 - i i 2 0 .
IO03
(25) Eddowes, M. J. Anal. Roc. 1989, 26, 152-154. (26) Kooyman, R. P. H.; Kolkman, H.; Van Gent, J.; Greve, J. Anal. Chlm. Acta 1988, 213, 35-45. (27) Bergveld, P. Blosensws Bldectron. 1991, 6 , 55-72. (28) Newman, A. L. Patent WO 88/09499. 1988. (29) Stanbro, W. D. Patent WO 88/08528, 1988. (30) Newman, A. L. Patent WO 90/02792, 1990. (31) Peareon. R. K. AIChESymp. Ser. 1985. 267. 42-45. (32) Betalllard, P.; Gardles, F.; Renauld, N. J.; Martelet, C.; Colln, B.; Mandrand, B. Anal. Chem. 1988, 60, 2374-2379. (33) Gardles, F.; Martelet, C. Sens. Acfuators 1989, 17, 461-464. (34) Valdes, J.; Wall, J. G.; Chambers, J. P.; Eldefrawi, M. E. John Hqpklns APL Tech. Dig. 1988, 9 , 4-10. (35) Newman, A. L.; Hunter, K. W.; Stanbro, W. D. Roc. Int. Meet. Chem. Sens., 2nd 1988, 596-598. (36) Keli. D. B.; Davey, C. L. Bksensors, a pracfkal approach; IRL Press: Oxford University, Oxford, U.K.. 1990; Chapter 5. (37) , . Dbale. J. W.: Downie. T. C.: Gouldlnn. C. W. Chem. Rev. 1989. 69, 36g-405. (38) Vermilyea, D. A. Acta Metall. 1953, 1 , 282-294. (39) Pethlg, R.; Kell, D. B. phvs. M .Bid. 1987, 32, 933-970. (40) Sarma. V. R.; Sliverton, E. W.; Vavies. D. R.; Terry, W. D. J. Blol. Chem. 1071. 246. 3753-3759. (41) Schwan, H. P.yFehs, C. D. Rev. Sci. Inst”. 1988, 39, 481-489. (42) Gobrecht, H. Lehrbuch der €xperimenfa@ysk, 7th ed.; Walter de Gruyter: Berlin, New York, 1978; Vol. 111, Chapter 3. (43) Weetall, H. H. I n Methods of Enzymology; Mosbach. K., Ed.; Academic Press: New York. 1976; Voi 44, Chapter 10. (44) Sonnenfeld, R.; Hansma, P. K. Science 1988, 232, 211-213.
RECEIVED for review September 27,1991. Accepted January 27, 1992.
Liquid Chromatographic Separation of Alkanesulfonate and Alkyl Sulfate Surfactants: Effect of Ionic Strength Daan Zhou and Donald J. Pietrzyk*
University of Iowa, Chemistry Department, Iowa City, Iowa 52242
The retention of alkanesulfonate and alkyl sulfate surfactants, which was determined on a reversed stationary phase as a function of mobile-phase ionic strength, Is consistent with a double-layer type interaction at the stationary-phase surface. Increasing the mobilephase lonlc strength not only increases retention but also Improves resolution because peak widths are dgnlficantly reduced. The type of cation provided by the ionk strength salt also enhances retention, reduces peak width, and improves rewlutlon. Lithium hydroxide is an ideal electrolyte for the separation of mutticomponent mixtures of aikanesulfonate and alkyl sulfate surfactants. When the c d m n effluent is passed through a postcolumn anion micromembrane suppressor, the conductivity due to the electrolyte is minimized and conductivity detection is sendive, yielding a detection limit of about 0.3 nmoi of injected anaiyte for a 3:l signaknoise ratio. Multicomponent alkanesulfonate and alkyl sulfate mixtures from C2to C,8 are baseline resolved by udng a moblleghase gradient whereby CH&N concentration increases and LlOH concentration decreases.
INTRODUCTION Typical anionic surfactants will contain hydrophilic (anionic) and hydrophobic centers, and both contribute to the surfactants physicochemical properties. Major classes of anionic surfactants are the long-chain alkanesulfonates (ASO,-), alkyl sulfates (AOS0,-), and the linear alkyl-
benzenesulfonates(LAS). All three are water soluble and are used widely in commercial products and processes where detergent action is required. The need to accurately determine their presence in these applications and in the environment, particularly at trace levels, is of growing concern. The development of analytical methodology for the determination of anionic surfactants has focused on the properties of either the hydrophobic or the hydrophilic portions of the surfactants. In general, classical procedures, such as precipitation, color formation, and color quenching involve a reaction between the anionic center and another reagent containing a positive center to produce a neutral product of low dis~ociation.’-~These methods, however, do not discriminate among individual compounds within homologous groups of anionic surfactants and, consequently, their main application is generally toward group determinations. Classieal separation strategies, such as planar methods, ion exchange, and solvent extraction, which often require reactions a t the anionic center, are also primarily suited to group separations. In contrast, modern, efficient separation methodologies including gas Chromatography (GC),9*4-7 high-performance liquid chromatography (HPLC),&’*and capillary electrophore~is~~ are strategies which have been successfully used to discriminate between members within the homologous or isomeric groups of AS03-, AOS03-, and LAS surfactanta. The hydrophobic center, the anionic center, or both are key surfactant structural features which influence retention and resolution. In GC anionic surfactant volatility is often a limiting factor and has been overcome through desulfonation procedures4or
0003-2700/92/0364-1003$03.00/00 1992 American Chemical Society
