Anal. Chem. 1986, 58, 2874-2876
2874
surface due the large area/volume ratio in the gel electrode, and (iii) possible advantages in automation and the exclusion of macromolecules. For the relatively large geometrical area electrodes used in this study (0.28 cm2), aliquots of ca. 20-40 pL were applied to fresh electrode surfaces for ca. 5 min. The electrodes swell somewhat as the solution is absorbed by the gel. Application of additional aliquots to the surface did not result in absorption of more solution or increased peak currents. For Fe(CN)t- solutions differential pulse voltammograms exhibited a single wave a t 0.196 V after subtraction of the background current which contained peaks attributed to oxidation of the surface of the graphite particles. A calibration curve of peak current vs. Fe(CN)64-concentration was constructed by using data collected from three batches of graphite/pacr gel, each having slightly different characteristics (i.e., degree of polymerization and cross-linking). Although electrochemical response varied somewhat because of variation of the polymer gel morphology, the calibration curve was linear over the concentration range 0.2-100 mM with a correlation coefficient of 0.9907 and an intercept of -80 PA. For the individual data sets obtained with the same gel, the correlation coefficients were 0.9938, 0.9960, and 0.9875. The significant feature of these data is the wide dynamic range for the solution entrapped in the gel electrode. It is anticipated that detection limits can be significantly lowered by minimization of background currents. Also by use of smaller electrodes, it should be feasible to analyze volumes of 1 FL or less. Thus this approach has promise for the analysis of small volumes containing amounts in the 100 nmol range or less. The technique was also applied to p-benzoquinone solutions (10% in ethanol), which gave a linear calibration curve over the concentration range 1-50 mM. For HzQ, however, very large oxidation currents were observed that did not give well-defined peaks. This phenomonen is under further study. Solutions of MV2+absorbed in the gel electrodes gave welldefined peak currents, but the dication rapidly diffused out of the swollen electrode limiting the analytical utility of the method. In this situation it will be necessary to carry out the voltammetric quantitation with the loaded gel electrode contacting the electrolyte solution in a thin-layer configuration.
CONCLUSIONS Polyacrylamide gel electrodes have been fabricated by use of relatively simple procedures that require only readily available, inexpensive materials. It has been demonstrated
that complex, multicomponent, electroactive interfaces can be constructed using ordinary pacr gels. Specifically, composite electrodes containing graphite particles, solid mediator (ferrocene), semiconductor particles (TiOz), and enzymes (glucose oxidase (2))have been shown to function as catalytic and photoresponsive gel electrodes. The possible analytical utility of these electrodes was demonstrated for the differential pulse voltammetric analysis of small volumes of ferrocyanide and p-benzoquinone solutions. Charge transport through the ferrocene-loaded pacr gels obeys the laws of simple linear diffusion over a wide time scale. The Cottrell slopes were not strongly dependent on the percent ferrocene in the electrode formulation a t loadings above ca. 10%. The absence of a simple finite volume limitation of the mass transport process is probably related by the large volume changes that take place upon electrolysis with these electrodes, but a complete understanding of the change transport process awaits further work. Registry No. pacr, 9003-05-8;TiOz, 13463-67-7;Fe(CN):-, 13408-63-4;graphite, 7782-42-5; p-benzoquinone, 106-51-4;ferrocene, 102-54-5.
LITERATURE CITED (1) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hili, H. A. 0.; Aston, W. J. HiQQinS, I. J.; Piotkin, E. V.; Sott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 5 6 , 667. (2) Lange, M. A.; Chambers, J. Q. Anal. Chim. Acta 1985, 175, 89. (3) Van Koppenhagen, J. E.;Majda, M. J. Nectroanal. Chem. 1985, 189, 379. (4) Osada, Y.; Hasebe, M. Chem. Lett. 1985, 1285. (5) Oster, G. K.; Oster, G.; Prati, G. J . Am. Chem. SOC. 1957, 79, 595. (6) Duonghong, D.; Borgarello, F.; Gratzel, M. J. Am. Chem. SOC.1981, 103, 4685. (7) Burgmayer, P.; Murray, R. W. J. Hectroanal. Chem. 1902, 135, 335. (8) Kolthoff, ,I. M.; Thomas, F. G. J. Phys. Chem. 1965, 6 9 , 3045. (9) Dunn, W.; Yosihiro. A.; Bard, A. J. J. Nectrochem. SOC.1981, 128, 222. (IO) Duonghong, D.; Ramsden, J.; Gratzel, M. J. Am. Chem. SOC.1982, 104, 2977.
Mark A. Lange James Q. Chambers* Department of Chemistry University of Tennessee Knoxville, Tennessee 37996
RECEIVED for review March 18, 1986. Accepted July 1,1986. This research was supported by grants from Martin Marrietta Corp. and the National Science Foundation (Grant No. CHE-8219210).
Fiber-optic Probe for Kinetic Determination of Enzyme Activities Sir: The knowledge of enzyme concentration of a sample solution is of considerable practical interest in clinical analysis, and numerous methods have therefore been proposed for assays ( I , 2). Most of these are kinetic, based on the measurement of changes in the concentration of a natural or synthetic substrate. Assays have mainly been performed in solution or on “dry reagent” phases (3). None of them, however, is suitable for in vivo application. Our interest in photometric and fluorometric enzyme activity determination ( 4 , 5 )along with that in fiber-optic sensors (6) resulted in the idea to determine enzyme activities via fiber optics in very small sample volumes with the aim to develop a method for an in vivo assay in blood and related biomatter. Small optical fibers have already been used for in vivo sensing
of pH (3,oxygen (8),or pH, oxygen, and C 0 2simultaneously (9) because fiber sensors have certain advantages over electrochemical sensors. Biocompatibility, small size, and the lack of reference cells appear to be the most attractive features. Arnold (IO) has recently described the immobilization of the enzyme alkaline phosphatase (together with light-scattering material) a t the end of a fiber-optic probe. When immersed into a substrate solution such as p-nitrophenyl phosphate, the second fiber sees the formation of yellow pnitrophenolate. This communication describes our experiments directed toward a continuous kinetic enzyme assay via optical fibers. In contrast to the method of Arnold (IO), the substrate is immobilized in this case, and the formation of the hydrolysis
0003-2700/86/0358-2874$01.50/0 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
2875
L
al
ferrule
u C
t
al V
m
E
0
1
Figure 1. Schematic of the optical arrangement.
d
product, which remains in immobilized form, is monitored.
.-cal> m
d
EXPERIMENTAL SECTION A schematic of the experimental setup is shown in Figure 1. Light from a xenon light source (L) of an Aminco SPF 500 fluorometer was focused into the end of a 100-pm single fiber (type SWCC TK/CF-100/140, from Showa Electric Wire and Cable Comp., Tokyo). After passing the optical coupler, OC (where fibers F1and F2are combined to one single fiber F), the exciting light of wavelength 465 nm was guided to a membrane with an immobilized enzyme substrate on it. The membrane was pulled over the ferrule of the fiber end (20 mm 0.d.) and fixed with an O-ring or epoxy. Green fluorescence of the membrane, which is almost zero in the absence of an esterase, and scattered blue light return through the same fiber. Fifty percent of the total returning light is coupled into fiber F, in OC. After the light passes a secondary filter adjusted to 530 nm, fluorescence is detected with a photomultiplier and its intensity displayed in the SPF 500 instrument. The membrane with immobilized substrates for cholinesterase and related carboxylesberases was prepared as follows: 1hydroxypyrene-3,6,8-trisulfonate (HPTS) was bound to a strong anion exchange membrane (Raipore R-1035, from RAI Research Corp., Long Island, NY) by electrostatic immobilization. The procedure is similar to the one described by Zhujun and Seitz (11). Esterification of HPTS with an acid chloride or anhydride gives the respective ester. In a typical experiment, a 1 X 1 cm piece of the membrane was immersed into a solution of 0.1 mg of HPTS (Lambda Chemie, Grottenhof-Str. 3, A-8053 Graz, Austria) in 5 mL of deionized water. After 1day, the yellow membranes were rinsed with water and acetone and treated for 3 h with ca. 1mL of the anhydrides of either acetic or butyric acid. The color changed from yellow to pale yellow, and fluorescence changed from green to violet after this treatment. Pieces of ca. 4 X 4 mm were cut, pulled over the ferrule at the distal end of the fiber, and fixed. All reagents were of usual analytical purity. The following enzymes were used: carboxylic ester hydrolase (enzyme catalog no. 3.1.1.1, from porcine liver), lipase (E.C. 3.1.1.3, from wheat germ), and acylase I (E.C. 3.5.1.14, from hog kidney), all from Sigma Chemie, Munich. All experiments were performed in a room thermostated to 23 "C.
RESULTS AND DISCUSSION Fatty acid esters of l-hydroxypyrene-3,6,8-trisulfonate (HPTS) have been shown to be useful substrates for a direct and kinetic assay of esterases (4). Their structures are given in the formula scheme. Three sulfonato groups make i t easy
substrate : a c e t a t e , R = CH3-CO butyrate , R = C,H,-CO
HPTS
HPTS phenolate anion
to immobilize the dye on an anion exchange membrane (11). Because it was found that immobilization of either the HPTS acetate or butyrate by immersing the ion exchange membrane into the respective methanol solutions of the substrates resulted in considerable nonenzymatic hydrolysis, an alternative approach was attempted that gave much better results: HPTS was first immobilized and subsequently acylated. This pro-
E
30 LO 50 time (sec) Flgurr 2. Increase In fluorescence intenslty with time when immobilized HPTS acetate is immersed into a pH 7.2 solution of various amounts of carboxylesterase. The figures refer to micrograms of enzyme per millillter of solution, and n.e. is the increase in signal due to non enzymatic hydrolysis. 10
20
cedure was found to give a membrane possessing almost no green fluorescence, i.e., all HPTS is present in esterified form. Enzymatic action results in the cleavage of the substrate to give immobilized HPTS which, because of i b low pK, of 7.0, in phosphate buffer of pH 7.2 is dissociated by more than 50%. Both free HPTS and ita phenolate fluoresce at 520 nm with quantum yields close to unity (12). The 460-nm absorption band of the phenolate was chosen in this work for the excitation because the band is more intense than the 415-nm band of the phenol species. Moreover, the fibers have better transmission for blue light than violet light, and the xenon lamp has a higher output at 460 nm than at 415 nm. Figure 2 shows how fluorescence increases when the tip of a fiber with the HPTS acetate membrane is immersed into solutions containing various concentrations of carboxylic ester hydrolase. A fresh piece of membrane, cut from a 2 x 2 cm membrane prepared as described before, was used in each experiment. The curves are typical for an enzymatic reaction in showing a rapid increase in signal per time in the initial phase and a slow-down of the signal change as the substrate gets more and more consumed. The initial slope varies linearly with enzyme concentration. S i m i i results were obtained with the other hydrolases used in this work. While there is agreement in the relative rates of enzymatic hydrolysis with the data published for cuvette experiments (4), one notes a decrease in the initial reaction velocity by a factor of 3-5. Since, on the other hand, the rate of nonenzymatic hydrolysis is very slow a t pHs below 7.6,there is no need for a correction to account for this side reaction at enzyme levels a t or above 20 Fg-mL-l. The rate of autolysis is distinctly smaller for the HPTS butyrate membrane. Unfortunately, this substrate is hydrolyzed very slowly by all three enzymes tested. Consequently, the HPTS acetate is a much better substrate despite the larger rate of nonenzymic hydrolysis. The sensitivity of the assay is limited by the rates of the enzymatic and the nonenzymatic reaction. It can be seen from Figure 2 that the limit of detection for carboxylesterase is at about 20 FgmL-' protein. Below this level the signal change per time becomes too small (in comparison with autolysis) to be of analytical use, unless a correction is made for the latter process, The results show the feasibility of a fiber optical determination of enzyme activities. The method is considered to have two decisive advantages over former optical methods:
2878
Anal. Chem. 1980, 5 8 , 2876-2879
I t allows a determination over distances of meters and in a rather small sample volume which, in principle, is limited only by the size of the fiber. It therefore provides a suitable means for invasive detection and determination of enzyme activities. Since assays can be performed in vivo, real-time probing becomes possible. The fluorescent dye, which, conceivably, may be physiologically harmful, remains in the immobilized state so that it does not present a risk in in vivo sensing. The acid part, in contrast, is released, but this is the “natural” part of the synthetic substrate, for instance, a fatty acid anion, phosphate, sulfate, or a sugar molecule such as glucose. Because the substrate is irreversibly consumed, the probe has a limited lifetime. Consequently, it does not lend itself to continuous monitoring, which is in contrast to known invasive sensors for pH and oxygen (9). Therefore, the device cannot be called a true sensor which, by definition, is able to indicate continuously and reversibly a chemical or physical parameter. The probe works irreversibly, so that its calibration may be more difficult than in the case of reversible sensors. Since, however, a single membrane can be used for several short-time measurements (with gradual degradation in performance as the background from hydrolyzed HPTS increases), calibration appears feasible by making an initial measurement using an enzyme solution of defined activity. If separate membranes are to be used, one has to make sure that each sensor of a lot responds in the same way. This new principle has been shown to work with carboxylesterases in this paper. Numerous other chromgenic or
fluorogenic substrates are known ( I ) . Once they can be immobilized on a solid support and provided their substrate properties are conserved, they appear to be applicable for in vivo sensing of the respective enzymes via the fiber-optic approach presented here. Registry No. HPTS (acetate), 85353-19-1;HPTS (butyrate), 85353-20-4;carboxylesterase, 9016-18-6.
LITERATURE CITED Guilbault G. G. Enzymatic Methods of Analysis ; Pergamon Press: Oxford. 1970. Methods of Enzymatic Analysis; Bergmeyer, H. U., Ed.: Verlag Chemie: Weinheim, Deerfield Beach, 1984. Walter B. Anal. Chern. 1983, 55, 498A-512A. Wolfbeis 0. S.; Koller E. Anal. Blochem. 1983, 129,365-370. Koller E.; Wolfbeis 0. S. Anal. Biochern. 1984, 143, 146-151. Wolfbeis 0. S. Trends Anal. Chem. 1985, 4, 184-188. Peterson J. I.; Goldstein S. R.; Fitzgerald R. V.; Buckhold D. K. Anal. Chern. 1980, 52, 864-869. Peterson J. I.; Fitzgerald R. V.; Buckhold D. K. Anal. Chem. 1984, 56, 62-67. Gehrich J. L.; Lubbers D. W.; Opitz. N.; Hansmann, D. R.; Miller, W. W.; Tusa, J. K.; Yafuso, M. IEEE Trans. Horned. Eng. 1986, 33, 117-132. Arnold, M. A. Anal. Chern. 1985, 57, 565-566. ZhuJun, Z.; Seitr, W. R. Anal. Chirn. Acta 1984, 160, 47-55. Wolfbeis, 0. S.; Furiinger, E.; Kroneis, K.; Marsoner, H. Z. Anal. Ch8m. 1983, 314, 119-124.
Otto S. Wolfbeis Analytical Division Institute of Organic Chemistry Karl Franzens University A-8010 Graz, Austria RECEIVED for review May 6, 1986. Accepted July 7, 1986.
Photothermal Quantitation of Nanogram Quantities of Coomassie Brilliant Blue Stained Proteins in Denaturing Polyacrylamide Gels Sir: Photothermal spectroscopies are a class of ultrasensitive indirect absorption measurements, in which the heat evolution accompanying light absorption is measured. The techniques all measure the local changes in sample refractive index caused by the heat evolution (I-3), as the expansion, deflection, or refraction of a laser beam. Photothermal techniques have been shown to provide superior sensitivity to direct absorption measurements in liquid chromatography (4-6)and thin-layer chromatography (7,8). In the crossed beam thermal lens experiment, a pump/probe configuration is used with the two lasers crossed, rather than collinear. This configuration is simpler to align than the more familiar collinear laser version. In a preliminary report we demonstrated that crossed beam thermal lens spectroscopy can be used to quantitate bovine serum albumin stained by Coomassie Brilliant Blue G250 in nondissociating polyacrylamide gel electrophoresis (9). We obtained detection limits of 1ng using He-Ne laser radiation to generate a thermal lens. The detection limits are about 2 orders of magnitude lower than the visual limit of detection in the same system. For separation purposes, nondissociating electrophoresis is often adequate. However, the characterization of a new protein often begins with an electrophoretic estimate of molecular weight using polyacrylamide gel electrophoresis of a denatured protein. The electrophoretic mobility of a protein depends on its net charge, molecular weight, shape, and the
rigidity of packing of the polypeptide chain. The contribution of these factors varies considerably with experimental conditions. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), the Laemmli procedure (IO), allows elimination of all the factors except molecular weight. In the present communication we apply the photothermal detection technique to the Laemmli SDS-polyacrylamide gel procedure. We describe experimental problems in the use of photothermal detection with SDS-polyacrylamide gel electrophoresis and p r o p e solutions to them. The detergent SDS causes problems in fixation, staining, and quantitation of proteins with dyes (11). Therefore, we compare the performance of Coomassie Brilliant Blue R250 and G250 in the presence of SDS in this application.
EXPERIMENTAL SECTION Polyacrylamide gel electrophoresis was performed by Laemmli’s dissociating discontinuous method (SDS-PAGE). All electrophoresis reagents were obtained from Sigma. Acrylamide and N,”-methylenebis(acry1amide) were recrystallized by Loening’s method (12). Other materials were used without further purification. Low molecular weight protein standards (BioRad), phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme were used to evaluate the procedures. Polyacrylamide gels (10% acrylamide) were cast in 1 cm thick slabs. The protein samples were loaded onto the wells 10 mm
0003-2700/86/0358-2676$01.50/0 0 1986 American Chemical Society
