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Langmuir 1988, 4 , 776-780
excite interband transition and mainly induces the local excitations such as the 0-Fe charge transfer, the enhancement of SO2adsorption in a-FeOOH systems should be related with the excited surface oxygens. Recent FT-lR examination22showed that SO2 reacts with the surface oxygen of a-FeOOH to produce S0,2--like species without (22) Mataumoto, A.; Kaneko, K. Abstract of 40th Colloid Interface Chemical Symposium; Kyoto, Japan, 1987; p 1A12.
photoillumination. It is presumed that the photoillumination accelerates the formation of the SOa2--likespecies with the use of an oxygen on the a-FeOOH surface. The photoenhanced SO2adsorptivity of a-FeOOH must be in connection with the phenomena in atmospheric environments such as acid rain formation and acceleration of atmospheric corrosion of iron by SOz. Registry No. SOz, 7446-09-5; FeO(OH), 20344-49-4; Ti, 7440-32-6.
Electrophoretic Fingerprinting and the Biological Activity of Colloidal Indicators B. J. Marlow* and D. Fairhurst Pen K e m , Inc., Bedford Hills, New York 10507
W. Schutt Department of Internal Medicine, Wilhelm-Pieck University Rostock, 2500 Rostock, GDR Received January 8, 1988. In Final Form: March 9, 1988
The electrophoretic mobility of colloidal indicator particles, contacted with human body fluids under physiological conditions, can be used to diagnose and follow the treatment of a variety of pathological conditions. Batches of the same types of particles, e.g., polystyrene latex microspheres, prepared under seemingly similar conditions, may or may not respond to these advantageously simple testa. This work describes the use of electrophoretic fingerprinting to discern the biological activity of two batches of polystyrene latex microspheres: one responsive to human body fluids and the other unresponsive.
Introduction At Rostock University in the Department of Internal Medicine, Schutt et al. have been carrying out extensive research on the application of cell microelectrophoresisto clinical diagnosis and treatment, a review of which is currently in preparation. They have shown that the electrophoretic mobility of indicator colloids, such as silicone oil droplets and polystyrene microspheres, can be used to diagnme and follow the treatment of disorders such as cystic fibrosis, fetal lung maturity, lung cancer, pneumonia, meningitis, and respiratory distress syndrome in newborns. Clinical studies have included some 600 patients. These disorders invariably cause a change in the composition of certain body fluids, e.g., serum, bronchoalveolar lavage, cerebrospinal fluid, and pharyngeal secretions from newborns. The change in body fluid causes a change in the electrophoretic mobility of indicator particles when contacted with the fluid. These body fluids are easily accessible, and the test can be clinically performed rapidly by lab technicians using automated electrophoresis equipment. The changes in the particular body fluid are manifest by a shift, relative to healthy patients, of the electrophoretic mobility of indicator particles that have been contacted with the body fluid. An example of this trend is shown in Figure 1. The figure shows five electrophoretic mobility distributions of polystyrene latex microspheres (0.20 fim) in a phosphate buffer and physiological strength saline. Under these conditions the microspheres had a mean electrophoretic mobility U, of -1.95 x lo* m2/(V.s). When these microspheres are contacted with the same concentration of serum from both healthy and cystic fibrosis patients the mobility shifts to -1.09 X 10-8 and -0.87 X lo* m2/(V.s), respectively. The shift in mobility of 20%
from healthy to afflicted patients is in excess of the experimental error of -2% for the apparatus used in this work (see below). Extensive clinical studies by Shutt et al. have confirmed a definite statistical trend. Schutt et al. have found that only certain types of indicator particles respond in their simple tests. Indeed, the response of different batches of the same type of particles to body fluids cannot be predicted. The objective of this note is to describe the differences in surface chemistry found between two batches of polystyrene latex microspheres used by Schutt et al.; one batch is responsive in these tests while the other is unresponsive. The differences are deduced by using the technique of electrophoretic fingerprinting developed at Pen Kem, Inc., and first applied tQ latex surfaces by Marlow, Rowell, and Morfesis.’+ The focus of this paper is to demonstrate the usefulness of electrophoretic fingerprinting in analyzing biologically active versus nonactive surfaces; it is not a definitive description of the role of surface chemistry and biological activity per se. The ability to determine biological activity of colloidal surfaces is both timely and important to such fields as hematology, neurology, pediatrics, clinical immunology, tumor immunology, pharmacology, and biophysics, thus, the impetus for this brief communication. Electrophoretic fingerprints or templates are three-dimensional representations of the mean electrophoretic (1) Marlow, B. J.; Fairhurst, D.; Oja, T. Abstracts; 60th Colloid and Surface Science Symposium, Georgia Tech., Atlanta, GA, June 15-18, 1986. (2) Morfesis, A. Ph.D. Thesis, University of Massachusetts, Amherst, MA, 1986. (3) Marlow, B. J.; Rowell, R. L.; Morfesis, A. Gordon Research Conference on Pol” Colloids, Tilton School, Tilton, NH, July 6-10,1987. (4) Marlow, B. J.; Rowell, R. L.; Morfesis, A. Abstructs; Division of Colloid and Interface Science, Fall ACS Meeting, New Orleans, LA, 1987.
0743-746318812404-0776$01.50/0 0 1988 American Chemical Society
Langmuir, Vol. 4, No. 3, 1988 I71
Letters
A/,,
heres La t ;e ;x (
-1.95 ' h
--1.09
Healthy Patient
.,
.n
Healthy Patient
-1.08
Cystic Fibrosis
-0.94
Cystic Fibrosls Patient
JW -, -4
-3
-2
%mZ/vI
-1
x
0
io*
Figure 1. Electrophoretic mobility distributions of polystyrene latex microspheresin phosphate-buffered physiologicalstrength saline compared to the same latex contacted with serum from two healthy patients and two carrying the cystic fibrosis gene.
mobility of a given colloid versus pH and conductivity. The fingerprint representa a surface, described by isomobility lines, over all pertinent electrochemical conditions. Analysis of electrophoretic mobility data in this manner is justified from a theoretical point of view, since all theories that treat the charging of colloidal surfaces in an aqueous environment have as variables potential, pH (when H+and OH- are potential determining), and conductivity.SyB The advantages of such a treatment are discussed below. The potential and pH represent thermodynamic or equilibrium values whereas the conductivity reflects the overall ionic transport properties of the media. Some may argue that the third variable should actually be ionic strength since this is proportional to ion concentration. However, double-layer theory does not support this notion. Invariably, for theories that deal with the electrostatic charging of surfaces in aqueous electrolytes, the effect of ion concentration is manifest through a parameter which can be shown to be proportional to the ratio of the inverse Debye length to total ion concentration. To a first-order approximation this term can be represented by conductivity. The pertinent variable, therefore, which reflects the total ion concentration and the overall ionic transport properties of the media is conductivity rather than ionic strength. This matter will be addressed in detail in a separate paper.
Experimental Section Methods. Two batches of polystyrene latex microsphereswere prepared under the same conditions by surfactanbfree emulsion polymerization using potassium peroxydisulfate (K2S2O8)as initiator. The latex batches were cleaned by serum replacement. The latices had a mean particle radius of 0.20 pm and were not chemically modified. Samplesare prepared for microelectrophoresisby placing three drops of an approximately 10% polystyrene latex microsphere dispersion in 600 mL of HPLC grade distilled water (J.T. Baker Chem. Co.). The colloid is then sampled automatically, and the electrophoretic mobility distribution, pH, and conductivity are measured. The sample is then automatically dosed with the (5) Healy, T.W.;White, L. R. Adu. Colloid Interface Sci. 1978, 9, 303-345. (6) Hunter, R. J. Zeta Potential in Colloid Science; Academic: New York, 1981; Chapter 7.
appropriate acid or base from a digital buret, resampled, and analyzed. All measurements were performed at 25 "C with the Pen Kem System 3000 Automated Electrokinetics Analyzer, described elsewhere.'+ In the present work, the pH is altered by using the supporting electrolyte (NaC1) conjugate acid (HC1) and base (NaOH). If, for example, one starts in distilled water and titrates with NaOH and then reverse-titrates with the HCl, the net effect is not only a change in pH but also the production of NaC1, i.e., an increase in conductivity. Thus, back-and-forth titration with the supporting electrolyte conjugateacid and base allows one to vary both pH and conductivityover all electrochemicalconditionsaccessible. The System 3000 performs this function automatically in a few hours. Data-Plotting Algorithms. Electrophoretic mobility data are collected as a function of two variables, namely, pH and conductivity. Collectivelythe mobility data can be represented aa a three-dimensionalorthogonalplot, where pH and conductivity represent the independent variables and mobility the dependent variable, giving the third spacial axis. Typically, 100-400 individual spacial points are used to produce the fingerprint. The data analysis is performed with software algorithms developed by Golden Software, Inc., Golden, CO. The data are first gridded to produce regularly spaced data in the x-y plane required for contour mapping and three-dimensional display. The gridding procedure uses Kringing, a technique that utilizes a regional variable theory and assumes an underlying linear variogram.10 The gridded data are then smoothed by using a cubic spline. Splining removes only jagged edges from the surface and does not 'curveWfit Thus, the surfaces, represented in this work, reflect the original data. Statistical analysis and curve fitting would, obviously,result in smoothing of the surface. Once gridded and splined the data can be represented as a three-dimensional plot (template) or as an isomobility contour plot (fingerprint).
Results The electrophoretic template (three-dimentional representation) and fingerprint (isomobility contour plot) of the batch of polystyrene microspheres unresponsive to body fluids are shown in Figures 2 and 3, respectively. The advantage of representing the data in this manner is that (7) Goetz, P. U.S.Patent 4154669, May 15,1979. (8) Goetz, P. Cell Electrophoresis; Proceedings of the Intemational Meeting, Roetock, German Democratic Republic, Schutt, W., Iumkmann, H., Eda.; Walter de Gruyter: Berlin, 1985. (9) Marlow, B. J.; Rowell, R. L. Energy Fuels 1988, 2, 125. (10)Ripley, B. D. Spatial Statistics; Wiley-Interscience: New York, 1981.
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Letters
is present, nor is there evidence to suggest an expandable layer at the ~ u r f a c e . ~The - ~ single acid site surface is characterized by an increasing negative mobility, as the pH increases, until a maximum value is attained, after which the mobility remains constant with pH. Also, this type of surface has no isoelectric point but approaches a zero mobility, or {potential, asymptotically as the pH is lowered. An example of the mobility versus pH at constant conductivity, SC, of 0.001 S/m or an NaCl concentration of =l X mol/L is shown in Figure 6 along with the predictions of the SASDM using the above parameters. A complete description of the analytical technique is in preparation for publication. Theory indicates that single-site surfaces are nonNernstian, Le., the slope of the t potential versus pH as { 0 # 59.1 mV.5v6 Thus, near the isoelectric point the surface is characterized by the absence of charge rather than equal numbers of positive and negative site^.'^-^^ Also, such a surface shows a monotonic decrease in mobility with increasing ionic strength that eventually levels off at high ionic strength, a feature predicted by the SASDM. For example, Figure 7 is a plot of the mobility of the unresponsive microspheres as a function of the logarithm of the conductivity compared to SASDM predictions using the parameters given above. This curve was produced by taking a cut of the surface shown in Figure 2 at a constant pH of 7.4, which is comparable to the pH used in the clinical studies of Schutt et al. The data of Figure 7 also provide evidence that Na+ or C1- adsorption does not occur at a pH of 7.4 on the unresponsive microsphere surface. The above conclusions regarding the surface chemistry of the unresponsive microspheres were deduced from cuts taken at constant conductivity and pH. As the conductivity increases the surface chemistry can be modified through specific adsorption of either co-ions or counterions. One notes from the fingerprint in Figure 3 that specific adsorption of cations occurs but only at low pH values. This results in a reversal in sign of the electrophoretic mobility, producing a region of positive mobility aad an additional isoelectric line. One can see from the major isoelectric line in Figure 3 that the isoelectric point increases from =4 at low conductivity to ~ 4 . at 8 high conductivity, supporting the notion of cation adsorption. The cut taken at a pH of 7.4, which is comparable to the physiological conditions used by Schutt et al., is unaffected by specific adsorption phenomena. Representing the electrophoretic data collectively in this manner and taking a cut from the fingerprint at low ionic strength produce a mobility dependence that results solely from the functional groups on the surface. The effects of cation (or anion) adsorption do not alter the,above conclusions about the functional groups on the surface but rather show that, under appropriate conditions, surface sites can adsorb cations. The electrophoretic fingerprinting technique is thus a powerful method to analyze the true functionality of a surface without the complicatingeffects of adsorption. The electrophoretic template and fingerprint of the batch of polystyrene microspheres responsive to body fluids are shown in Figures 4 and 5, respectively. These microspheres have a surface which can be described by a two-site dissociation model TSDM.5,6J4-19 This model
-
e ’ Figure 2. Electrophoretictemplate of polystyrene latex microspheres unresponsive to clinical studies. The pH is altered with NaOH and HCl. Isoelectric tinea /I
\
U e (m/s/V/m)
x E6
PH
Figure 3. Electrophoretic fingerprint of polystyrene latex microspheres unresponsive to clinical studies. The pH is altered with NaOH and HC1. Isoelectric lines represent the zero mobility contours; (- - -) positive mobility contours; (-) negative mobility contours. cuts can now be taken, from the isomobility plots, at strictly constant conductivity, or pH, for comparison with theoretical calculations. Conventionally, at low or moderate conductivities, a pH versus mobility plot will be taken along a diagonal of this surface because of changing conductivity. This technique obviates this pitfall. The regions blanked of data in these figures are experimentally inaccessible. For example, one cannot have, simultaneously, a high or low pH while the conductivity of distilled water (=lX S/m) is retained. By taking cuts at true constant conductivity and pH, from the surfaces shown in Figures 2 and 3, we are able to show that the unresponsive microspheres had a surface which can be described by a single acid site dissociation model (SASDM),5*6 the acid site being characterized by a pK, (-5) similar to that of a carboxylic acid. The number of acid sites at the shear plane is -5 X 10l2sites/cm2 or an area per charge site of 2000 A2. This is a low charge density compared to most polystyrene latices,ll which have a shear plane area of 100-1000 A2/unit charge. There is no evidence to suggest more than one type of acid group (11) Bangs, L. B. Uniform Later Particles, 2nd ed.; Seragen Diagnostics: Indianapolis, IN, 1985.
(12) Arp, P. A. J. Colloid Znterface Sci. 1983, 96,80. (13) Smith, A. L. Dispersions of Powders in Liquids, 3rd ed.; Parfitt, G. D., Ed.; Applied Science: NJ, 1981. (14) Levine, S.; Smith, A. L. Discuss. Faraday SOC. 1971, 52, 290. (15) Smith, A. L. J. Colloid Znterface Sci. 1976, 55, 525. (16)Healy, T. W.; Yates, D. E.; White, L. R.; Chan, D. J. Electroanal. Chem. 1977,80, 57.
Langmuir, Vol. 4, No. 3, 1988 779
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SC=O c c l S / m I m 0
R ~ (pLo n 8 i v e
E
-4-
Unresponsive
-8, 3
5
7
9
11
PH
Figure 6. Electrophoretic mobility as a function of pH taken from a cut of constant conductivity (0.001 S/m) from the electrophoretic fingerprints of both the responsive (@) and unreslatex microspheres. The data for the ponsive (w) polystyrene . . ~ Enresponsive microspheres are compar-d to calculations from the SASDM (---).
I
-! m
-
Figure 4. Electrophoretictemplate of polystyrene latex microspheres responsive to clinical studies. The pH is altered with
pH=l
4
l 01
Responsive
NaOH and HCl. N
E -4 soe elect tic Line
Ue (m/s/V/m)
x E8
3
Unr e s b o n s i v e
-43
4
5
6
7
8
9
10
11
PH
Figure 5. Electrophoreticfingerprint of polystyrene latex microspheres responsive to clinical studies. The pH is altered with NaOH and HC1. Isoelectric lines represent the zero mobility contour; (- - -) positive mobility contours; (-) negative mobility contours.
would, in practical terms, apply to a surface having groups whose dissociation can be described by either of three mechanisms: (i) the surface possesses two distinct (acid) dissociation sites; (ii) the surface has a single amphoteric group which may dissociate to yield a H+ or take up a H+, similar to oxide surfaces; and (iii) the surface is zwitterionic and possesses two distinct groups, one that can accept H+ and one capable of dissociating to yield a negative site. Since the responsive microspheres show a well-defined isoelectric line (see Figure 6), one can rule out mechanism i, where the surface will not exhibit an isoelectric point. Also, it is unlikely that a normal polystyrene surface is zwitterionic, thus ruling out mechanism iii. Also, apparent from Figure 6, a t low ionic strength the responsive microspheres show characteristics of an amphoteric surface, i.e., mechanism ii, the surface group being more than likely OH. Such a group can easily be produced through hydrolysis of sulfate groups.20*21 (17) Hartley, G. 5.;Roe,J. W. T r a m . Faraday SOC.1940,36, 101. (18)James, R.0.; Parks,G. A. Surface Colloid Sci. Matijevic, E., Ed.; Wiley-Interscience: New York, 1980; Vol. 11. (19)Rendall, H. M.;Smith, A. L. J . Chem. SOC.,Faraday Trans. 1 1978, 74,1179. (20)Van de Hul, H.J.; Vanderhoff, J. W. Br. Polym. J . 1970,2,121.
(21)Yates, D.E.;Ottewill, R. H.:Goodwin, J. W. J. Colloid Interface Sci. 1977,62, 356. (22)Zukoski, C.F.;Saville, D. A. J . Colloid Interface Sci. 1986,114, 32. (23)Zukoski, C.F.;Saville, D. A. J . Colloid Interface Sci. 1986,114, 45.
Langmuir 1988,4, 780-781
780
sites rather than the absence of charge. From this analysis one can conclude that, under physiological conditions, the unresponsive microspheres surface will have predominantly negative electron-donor sites whereas the responsive microspheres will have both negative donor sites, positive acceptor sites, and, in addition, sites containing adsorbed chloride ions. Both surfaces will also contain neutral “backbone” sites. Although the data presented are not definitive in determining why one batch of polystyrene microspheres is responsive to body fluids
while the other is not, it clearly shows that surface chemistry plays a key role in the biological activity of indicator particles and the power of electrophoretic fingerprinting in discerning the chemistry of biologically active versus nonactive surfaces. We believe that this simple technique can be an important analytical tool to help unravel the complexities surrounding biological activity of colloidal surfaces. Registry No. Polystyrene, 9003-53-6.
Notes Oxidation of Alcohol Monolayers by Chromic Acid
I
i51 \ Z
Jamil Ahmad* and K. Brian Astin*
E
c
Department of Chemistry, University of Bahrain, P.O. Box 1082, Bahrain
25t+ \
Received December 8, 1987. In Final Form: January 28, 1988
Reactions in monolayer assemblies provide the opportunity to examine the reactivity of molecules constrained to the plane. This environment can produce novel effects; e.g., we recently reported that the cyclization of the monoterpenoid alcohol nerol could be controlled by compression of the alcohol fi1m.l Conformer selection could be induced, since the coiled conformers required for cyclization could not be formed in highly compressed films. In the present study we have examined the kinetics of the surface acid dichromate oxidation of 1-phenyl-l-hexadecanol (la) and its deuteriated analogue 1-[2H]-1phenyl-1-hexadecanol (lb) to 1-phenyl-1-hexadecanone(2) using high-performance liquid chromatography (HPLC) for analysis of reactant and product. As noted by Valenty? this provides more reliable kinetic data than measurements of surface pressure, surface potential, etc.
-ii- 0 2
The experiments were performed on a modified Langmuir trough, consisting of a thermostated multicompartmental PTFEtrough with a Wilhelmy plate balance? The subphases, water and acidified sodium dichromate solution (0.10 M in 2% H2S04), could be confined to different compartments and the monolayers transferred from one to the other with minimal mixing. In a typical experiment M solution of the alcohol in hexane (la 20 pL of a 2 X prepared by the sodium borohydride reduction of 2, lb by sodium borodeuteride reduction of 2, >99 atom % D) was spread on water and compressed to 18 mN m-l (for ex(1) Ahmad, J.; Astin, K. B. J. Am. Chem. SOC.1986, 108, 7434. (2) Valenty, S.J. J. Am. Chem. SOC.1979, 101, 1. (3) Fromherz, P.Rev. Sci. Instrum. 1976, 46, 1380.
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\
\
t
\
0.4
-
0.6 o / ( n m ‘ m o l e c0.8 ule’)
Figure 1. a-u isotherm measured at 25 O C with a Wilhelmy balance (initial f i i area 170 cm2,fiial area 80 cm2,compression rate 24 cm2 min-’).
panded films R was
