1088
G. STONER AND S. SRINIVASAN
Adsorption of Blood Proteins on Metals Using Capacitance Techniques
by G. Stoner Department of Materials Science, School of Engineering and Applied Science, University of Virginia, Charlottesville, Virginia $8901
and S. Srinivasan Electrochemical and Biophysical Laboratories of the Vascular Surgical Services, Department of Surgery and Surgical Research, State University of N e w York, Downstate Medical Center, Broo?dyn, N e w York 11303 (Received September 17, 1080)
A differentialcapacity method was applied to the measurement of adsorption of proteins on solid metal electrodes. The differential capacity is measured by observing the square-wave current output response of two working electrodes in series to a small triangular voltage input. The double-layer capacity is then recorded as a function of electrode potential by a slow potential scan input into the potentiostated circuit. Capacity curves are first compared on mercury, gold, and platinum in pure solutions with reported literature values obtained by electrocapillary and ac bridge techniques. The coverages (0) of the amino acids glycine and tryptophan and the blood proteins fibrinogen, thrombin, and Hageman factor on mercury and platinum are then computed for various concentrations using the Frumkin equation, which relates 8 to the measured capacity. Glycine and Hageman factor were found not to adsorb appreciably on mercury or platinum whereas tryptophan, fibrinogen, and thrombin all adsorbed appreciably on both mercury and platinum. The adsorption results on mercury are in agreement with previous work using electrocapillarymethods, Introduction Over the last twenty years, considerable evidence has been accumulated to show that the interfacial reaction of thrombosis on the blood vessel wall and on prosthetic materials depends on the electrochemical characteristics of the solid-solution interface. 1-4 Thrombosis on conducting materials is found to be accelerated at potentials above 100 mV nhe and inhibited at negative potentials. This reaction is thus probably triggered by the interaction of one or more of the blood coagulation factors on the blood vessel wall or on the prosthetic materials at the more positive potentials. One of the characteristics of the adsorption of species on conducting surfaces from electrolytic solutions containing the adsorbates is its potential dependence. Thus, in the present work, the adsorption of some amino acids (the basic units of proteins) and some blood coagulation factors was determined on metal electrodes as a function of potential. One of the methods of obtaining information on adsorption of species on electrodes is by a determination of the capacity at the metal-solution interface as a function of potential. Bridge methods have been mainly used for capacity measurement^.^-' While these methods have proved useful for a determination of the capacity on mercury, several problems have been encountered in the extension of these methods to solid electrodes, these include time and frequency variation of capacitance due to unevenness of surface, adsorption pseudocapacity, dielectric relaxation, and impurities in solution and on the electrode.8 A sine-wave voltage, superimposed on a triangular potential sweep, coupled with an ac impedence bridge has been used by Breitero to The Journal of Physical Chemistry
measure capacitance. Recently, Gileadi and Tshernikovski1° have developed an instrument for the measurement of capacities across solid electrode-solution interfaces. In this method, a triangular wave in which the frequency and amplitude could be varied is superimposed between two small electrodes which are maintained at the same dc potential, and the amplitude of the resulting square wave is a measure of the capacity across the solid-solution interface. Thus, by making the measurements with varying dc potentials of the test electrodes it is possible to obtain the capacity of the electrode as a function of potential. The present work reports the following series of experiments, a few of which were carried out as a check of the method of Gileadi and Tshernikovksi and the remainder of which were done with the purpose of obtaining adsorption characteristics of some amino acids and blood coagulation factors. 1. Determination of the capacity-potential relation (1) P . N. Sawyer, “Biophysical Mechanisms in Vascular Homeostasis and Intravascular Thrombosis,” Appleton-Century-Crofts, New York, N. Y., 1965. (2) P. N. Sawyer and S. Srinivasan, A m e r . J. Surg., 114,42 (1967). (3) S. Srinivasan and P. N. Sawyer, J . Advan. Med. Inatrum., 3, 116 (1969). (4) G. Stoner and L. Walker, J . Bwmed. M a t . Res., 3, 645 (1969). (5) D. C. Grahame, Chem. Rev., 41,441 (1947). (6) D. C. Grahame, J . Amer. Chem. SOC.,63, 1207 (1941); 68, 301 (1946); 71, 2975 (1949). (7) K. Muller, Ph.D. Thesis, University of Pennsylvania, 1965. (8) S. D. Argade and E. Gileadi, in “Electrosorption,” E. Gileadi, Ed., Plenum Press, New York, N. Y., 1967, p 87. (9) M. W. Breiter, Electrochim. Acta, 7, 533 (1962). (IO) E. Gileadi, N. Tshernikovski, and V. Amdursky, Proceedings of 19th Meeting of C.I.T.C.E., Detroit, Mich., 1968.
ADSORPTION O F BLOOD PROTEINS
1089
ON n/IETAL
on mercury electrodes in 0.1 m NaCl in 1N NaOH for comparison of the results on the liquid metal with those of the classical work of Grahame,5v6who used bridge techniques. Experiments were also carried out in 0.1 N HC1 for which there are the recent data of Bockris, Gileadi, and 2 . Determination of the capacitance-potential relations on the solid metals: Au and Pt in M HC104. These metals were selected because of the fact that it is possible to work on them over a wide range of potential in which there are no competing reactions such as hydrogen or oxygen adsorption, metal dissolution, etc. The HC104 electrolyte was chosen because there is the recent work of Argades which may be used for confirmatory purposes. 3. Determination of the capacitance-potential relations on Hg and on Pt in 0.1 M NaCl containing varying concentrations of tryptophan and glycine. The reason for selecting these amino acids is that they are basic amino acids in blood protein, and tryptophan is strongly adsorbing on mercury while glycine is not.ll Hg is the simplest metal to work with and its surface is reproducible. Platinum was selected for the reason given above. I n addition, the capacity measurements were made on mercury in 0.1 M NaCl solution containing fibrinogen, thrombin, and Hageman factor. These compounds are actively involved in the intrinsic blood coagulation sequence.12 From the capacity-potential relations, the coverage-potential relations of the adsorbates typtophan, fibrinogen, and thrombin were obtained, according to the theory of Frumkin.13
Experimental Section 1. Electrical Components. The capacity meter, designed and developed by Gileadi and Tshernikovski, has been adequately described in the literature.1° The measured capacity in the form of a square wave was monitored on a Tektronix 546 storage oscilloscope. The magnitude of the capacity was recorded as a function of potential on a Hewlett-Packard Model 2D X-Y recorder. The potential of the working electrodes was varied using an Elron CP-1 potentiostat and Elron CHP-1 function generator. 2. Cells, Electrodes, and Solutions. The present method is most accurate when the series (solution) resistance between the two working electrodes is very small. Thus the distance between the electrodes was kept at a minimum by using a Teflon disk containing two concentric mercury electrodes (Figure 1) or two sputtered film electrodes (Figure 2) in the case of solid metals. I n alkaline and neutral solution, calomel reference electrodes were used and in acid solutions with the solid metals, reversible hydrogen electrodes were used. The solutions were prepared with conductivity water ( p > 2 X lo6) and reagent grade chemicals. The mercury was triple distilled (Bethlehem Instruments) and the sput-
Figure 1. Cell for measurement of differential capacity on mercury.
VITON O-RING I
LGLASS STOP-COCK
Figure 2. Cell for measurement of differential capacity on solid metals.
tered electrodes were prepared in a manner described e1~ewhere.l~Solutions of the protein fibrinogen and Hageman factor were prepared as described elsehere.^,'^ Pure bovine thrombin was obtained through the courtesy of W. Seegers. (11) G. Stoner, J. Bwmed. Mat. Res., 3, (1969). (12) W. Seegers, “Blood Clotting Enzymology,” Academic Press, New York, N. Y., 1967. (13) A. N. Frumkin and B. B. Damaskin, “Modern Aspects of Eleotrochemistry,” No. 3, J. O’M. Bockris and B. E. Conway, Ed., Butterworth and Co. Ltd., London, 1964. (14) J. O’M. Bockris, B. D. Cahan, and G. E. Stoner, Chem. Inst., 1, 273 (1969).
Volume 74, Number 6 March 6,i 9 Y O
G. STONER AND S. SRINIVASAN
1090
-
~-
VOLTiiGC SIGNAL 5-10 mY
4
PURPOSE
ELECTRODE RESPONSE
TO MEASURE DOUBLE LAYER RESPONSE CAPACITOR IS AN INTIGRATING CIRCUIT PRODUCING A SOUARE WAVE CURRENT OUrPUT WHOSE AMPLITUDE IS A MEASURE OF THE DOUBLE LAYER CAPACITY TO RECORD CAPACITY AS A CONTINUOUS FUNCTION OF POTENTIAL GOING ANODIC AND CATHODIC AMPLITUDE OF SQUARE WAVE IS RECORDED ON X-Y RECORDER AS A FUNC-
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15-20 MIN
-4TlON
OF POTENTIAL
MERCURY IN O.IM HCP
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1 2I 1 >
ELECTRODE WTENTIAL
3. Procedure. The capacity was recorded in the anodic and cathodic branches of a slow triangular potential scan and the criteria for an acceptable measurement were (i) suitable square wave form was obtained by varying the frequency of the triangular wave input; (ii) the lack of any hysteresis in the recorded capacity in the anodic and cathodic branches of the curve was achieved by reducing the overall scan rate to about V/sec. (Since the low range of the Elron instrument is V/sec, a 1/10 potential divider was used across the input of the potentiostat.) These procedures are illustrated in Figure 3.
Results 1. Comparison with Other Methods. (i) Measurements on Mercury. Capacity-potential curves in 0.1 M NaCl and 1 M NaOH are compared with the work of Grahame5v6in Figure 4 and for 0.1 M HC1 with h!Iuller7
I
.2
I
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1.0 1.4 POTENTIAL vs A H E
.6
1.8
Figure 5. Capacity as a function of electrode potential in 0.1 M HC1 on mercury. I
I
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SCAN RATE IO-^ v/sec.
26
ANODIC CATHODIC
-
PRESENT
-
in Figure 5. The above authors used ac bridge and electrocapillary methods. (ii) Measurements on Solid Metals. Figures 6 and 7 compare the respective capacity values on platinum and gold (in the neighborhood of the potential of zero charge) with the ac bridge work of Argade8 in dilute HClOa solutions. The values of the capacity are higher
MERCURY SCAN RATE 2 x
t
V/sec.
GRAHAME"-
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POTENTIAL vs RHE
Figure 6. Capacity as a function of electrode potential in dilute HClOb solutions on platinum.
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12L
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b
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1.18
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POTENTIAL vs N.C.E.
Figure 4. Capacity as a function of electrode potential in 1 M NaOH and 0.1 M NaCl on mercury. The Journal of Phyaical Chemistry
in the present work (probably due to differences in absolute electrode area or roughness factor) ; however, the shape of the curves and the characteristic minima are comparable. 2. E$ect of Various Blood Proteins and Relevant Amino Acids on Capacity-Potential Curves. (i)Measurements on Mercury. Capacity-potential curves are shown in Figure 8 for the amino acids glycine and tryptophan. Figure 9 shows the effect of the protein thrombin.
1091
ADSORPTION OF BLOOD PROTEINS ON METAL I
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Figure 9. Capacity as a function of potential in solutions of thrombin on mercury.
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a third blood protein, Hageman factor (Factor XII), did not lower the base (0.1 M NaC1) curve.
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Discussion I , Evidence for Using Technique of Gileadi and Tshernikovski for Measurments of Double-Layer Capacities on Metals in Solution. (i)Experiments on Mercury. Ex-
16-
-.2
-1.0 -1.4 -1.8 POTENTIAL AGAINST N.C.E.
-.6
-2.2
Figure 8. Capacity as a function of potential in glycine and various concentrations of tryptophan on mercury.
(ii)Measurements on Platinum. Capacity-potential curves for glycine and various concentrations of tryptophan are given in Figure 10. The effects of two proteins which are active in the blood clotting sequence, fibrinogen and thrombin, on the differential capacity are shown in Figures 11 and 12. Four concentrations (0.1, 1, 10, and 100 times physiological concentration) of
tensive work has been done on this metal using bridge technique^.^^^ The present measurements of the capacity as a function of potential in 1M NaOH, 0.1 M NaC1, and 0.1 M HC1 (Figures 4 and 5 ) are in good agreement with the corresponding C-T' relations obtained by other investigators using bridge techniques. This agreement lends considerable support to the method of Gileadi and Tshernikovski, whose technique has several advantages over the bridge technique in that (a) the method is simple and more rapid; (b) the capacity is readily recorded while the potential is varied linearly with time a t any desired rate; in this way by choosing the appropriate sweep rate, the effect of impurities in solution could be minimized; (c) the method can be used both on liquid Volume 74, Number 6 March 6, 1070
1092
G. STONER AND S. SRINIVASAN
and solid metals; and (d) bridge methods are for bridge techniques; it is necessary to use very small electrodes. Though in the present method there are advantages of small electrodes, measurements are still possible on electrodes of large areas. (ii) Experiinents on Gold and Platinum. The present results on platinum and on gold in HClOI are comparable with those of earlier work using bridge techniques. However, one observes a shift in the C-V curves of the present work. Higher capacities, recorded a t any potential in this work, may be due to the differences in the roughness factors of the electrodesthe capacities are represented per unit geometric area of the test electrode. 1. Method for the Calculation of the Coverage of Proteins as a Function of Potential from the CapacityPotential Plots. There are two possible approaches to obtaining information on coverage as a function of potential from the capacity-potential plots. I n one, which is a tedious procedure but perhaps more accurate, the C-V curve is integrated twice with respect to the potential (which gives a plot of the surface tension us. potential) followed by a differentiation of the surface tension with respect to the activity of the component whose adsorption behavior is to be ascertained. For this method, it is necessary to know the potential of zero charge (which is necessary to obtain the constant of integration of C us. V ) and also the surface tension a t any one potential which is necessary to evaluate the second integration constant. These constants are not readily available. Thus, the alternative method was chosen to obtain the coverages of the proteins as a function of potential of the test electrode. Here, the empirical equation proposed by Frumkin13 Ce = Co(l -
e)
+ eC,=l
which relates the measured capacity Ce to the degree of coverage (e) is used. Co and Ce..1 represent the capacities when 0 = 0 and 0 = 1, respectively. Though this equation is approximate, it has proved to be valid in the determination of adsorption of methanol on platinum from capacity measurements; there was good agreement in the coverage-potentials relations using this method and a transient technique.ls In order that this equation may be applied it is necessary to know COand Ce=i. The first, CO,is obtained from the capacitypotential relations in control solutions with no adsorbate. To obtain CeEl it is necessary to carry out the C-V measurements varying the concentration of adsorbate in solution and make the assumption the COS1 is the value of the minimum capacity for the highest concentration of adsorbate in solution. Generally one may observe a plateau in the region corresponding to the capacitance minimum (Figures 9-12). In the case of highly adsorbable materials, the minimum of the C-V plots may occur a t fairly low concentrations. A problem with this method will be encountered if the capacity The Journal of Physical Chemistry
-0
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Figure 11. Capacity m a function of potential for various concentrations of fibrinogen on platinum.
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a
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Figure 12. Capacity as a function of potential in solutions of thrombin on platinum.
in the presence of the adsorbate exceeds that on the absence of the adsorbate over certain potential regions. I n the present work, this was found only on mercury in solution containing tryptophan a t fairly anodic and cathodic potenitals. Even in these cases, it is possible to obtain coverage information in the region where this anomaly does not exist. 8. Adsorption Characteristics of Tryptophan on Mercury and on Platinum. Due to the fact that the capacity-potential curves on mercury in 0.1 M NaCl containing tryptophan (in varying concentration) intersects the control C-V curve (with no tryptophan), it is possible to determine the 8-V relation only over a short potential region (Figure 13). The adsorption a t any potential is markedly dependent on concentration. There is a steep fall in the coverage a t highly anodic M potentials. The adsorption maximum for concentration of tryptophan in solution occurs a t a potential of -0 V us. sce. (15) M. W. Breiter, EEectrochem. Acta, 7 , 633 (1962).
1093
ADSORPTION OF BLOOD PROTEINS ON METAL I
The adsorption of tryptophan on platinum from 0.1 M NaCl solution containing varying concentrations of tryptophan shows the typical type of parabolic 8-V behavioP for organic compounds on solid electrode
1
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Figure 16. Fractional coverage of thrombin as a function of potential on platinum. 1.8
1.6
1.4 1.2 1.0 0.8 POTENTIAL AGAINST N.C.E.
0.6
0.4
0.2
Figure 13. Fractional coverage of tryptophan as a function of potential on mercury.
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Figure 17. Fractional coverage of fibrinogen as a function of potential on platinum.
POTENTIAL vs N.C.E.
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(Figure 14). There is again a marked concentration dependence of adsorption. There appears to be no shift in the potential of maximum adsorption (-0 V vs. sce) with concentration of tryptophan. As on mercury, there is strong adsorption of tryptophan on platinum. 4. Adsorption of Thrombin on Mercury and on Platinum. Only one concentration of thrombin was used in the studies on mercury. The assumption was made that the coverage is unity in the minimum of the C-V relation on mercury in 0.1 M NaCl a t this concentration of thrombin. The results show that there is a significant adsorption of thrombin over a considerable range of potential (Figure 15). The adsorption maximum is quite cathodic (- 1,lV 11s. sce). The results of this work are in fair agreement with coverage data obtained from electrocapillary data.*l (16) E. Gileadi, B. T.Rubin, and J. O'M. Bockris, J. Phys. Chem., 69, 3335 (1965).
Volume 74, Number 6 March 6 , 1070
G. STONERAND S. SRINIVASAN.
1094 The shapes of the coverage-potential relations for thrombin in Pt show an unusual type of behavior-more than one maximum (Figure 16). Evidence was obtained for full coverage here at the highest peak because the same minimum capacity was obtained for the two higher concentrations of thrombin in solution. There is no shift in the position of the maximum with change of concentrations of thrombin in solution. The potential a t the highest peak is about 0.1 V vs. sce. The region of adsorption is quite extensive (-1 V). 6. Adsorption of Fibrinogen on Platinum. Fibrinogen plays an important role in the clotting mechanism.” Its adsorption is quite strong even a t concentrations which are less than physiological (maximum adsorption is observed a t 0.05-0.1of physiological concentrations). Parabolic 8-V relations are obtained (Figure 17). The potential of maximum adsorption appears to be independent of concentration and has a value of about 0.4 V. Adsorption is significant even at low concentrations of fibrinogen over a potential range of nearly 1V. The fall in adsorption at potentials cathodic to the potential of maximum adsorption (V,) is steeper than that a t potentials anodic to V,. 6 . Nonadsorbability of Hageman Factor. It is interesting to note that the capacity-potential relation on platinum is identical in the presence and in the absence of Hageman factor, which is the blood component initiating the blood coagulation mechanism. This important result indicates that Hageman factor does not trigger the reaction by an adsorption mechanism. This was confirmed by electrocapillary measurements on mercury in 0.1 M NaCl with and without Hageman factor in solution.
T h e Journal of Physical Chemistry
Conclusions The following conclusions may be reached from the present work. (i) The capacitance meter developed by Gileadi and Tshernikovski is a satisfactory instrument for capacity measurements on mercury and on solid metals. (ii) Tryptophan adsorbs strongly on mercury as well as on platinum. The coverage potential relations are quasi-parabolic. (iii) The coveragepotential relations of thrombin on platinum show more than one maximum. Thrombin is also a strongly adsorbing compound, though st more negative potentials. (iv) Fibrinogen shows the adsorption characteristics of simple organic compounds (e.g., hydrocarbons, alcohols). Maximum adsorption occurs a t a potential of 0.4 V vs. sce. (v) Hageman factor does not adsorb on mercury or platinum. It is possibly activated by a collision with surfaces of certain (positive) l8 charge characteristics.
Acknowledgments. The authors wish to thank Professors A. Catlin, E. Gileadi, and P. N. Sawyer for valuable discussions, suggestions, and encouragement of this work. Financial support from the National Institute of Dental Research, Grant DE-2111-05 (for G. S.) and from the Artificial Heart Program, National Heart Institute, National Institutes of Health, Contract No. PH43-68-75 (for S. S.) is gratefully acknowledged. S. S. is the recipient of a Career Scientist Award from the Health Research Council, City of New York, Contract No. 1542. (17) K. Laki, “Fibrinogen,” Marcel Dekker, Inc., New York, N. Y., 1968. (18) L. Vroman, Thromb. Diath. Haemorrhag., 10,446 (1963).
