Tissue bioelectrode for eliminating protein interferences - Analytical

Wang, Li Huey. ... Sarah Kirwan , Gaia Rocchitta , Colm McMahon , Jennifer Craig , Sarah Killoran , Kylie O'Brien , Pier Serra , John Lowry , Robert O...
1 downloads 0 Views 409KB Size
390

Anal. Chem. 1991, 63,398-400

The use of ultramicroelectrodes, now proven for the measurement of Ni(I1) concentrations in solution, was applied to the measurement of Ni(I1) in an individual cell of a cell culture of H4-II-C3 rat hepatoma cells. Cells in this particular cell line have an approximately spherical shape with a diameter nearing 10-15 pm. Thus the length of the sharpened/electroactive tip, which is plated with TMHPP, should be somewhat less than this dimension. The cell culture was grown directly on glass plates in Dalbecco‘s modified Eagle medium at 37 “C in a 10% C 0 2 atmosphere. The media was drawn off, and Tris buffer M Ni) was (containing 3% BSA, 2 mM glucose, and 1 X added. The cells were soaked in this Tris buffer solution for 45 min in order to allow Ni uptake. Following the uptake time, this Tris buffer was withdrawn and a fresh Tris buffer solution that did not contain Ni(I1) was added. Cells grown on glass slides in a Petri dish were placed on the inverted microscope, and the electrode was implanted by using the micromanipulator. When the electrode pierced the cell wall, the timing was initiated. The electrode was left in place for the desired preconcentration time. At the end of this time period the electrode was extracted, rinsed with deionized water, and placed in a 0.1 M NaOH solution for the determination of Ni(I1). The current from this analysis relates to the concentration of unbound Ni(I1) in the cell. For a 2-min preconcentration time, if the measurement was taken after the M Ni(I1) cell culture was subjected to a buffer with 1.0 x for 45 min, the resulting current represented a (1.0 f 0.1) X M Ni(I1) concentration in the cell. Total nickel accumulation, for conditions which were identical with those employed with the microelectrode studies, was determined by using liquid scintillation counting of 63Ni. The calculated nickel accumulation was 10 pg/million cells. Assuming the cells have a spherical shape with a radius of 7 pm, the total concentration of accumulated nickel, bound and unbound, in each cell would be 1 X M. Therefore, in a single rat hepatoma cell after 45 min, the total nickel accumulation was 0.01 pg and only 6% (0.6 fg) of this amount was unbound, as determined by using the ultramicroelectrode and differential-pulse voltammetry. After 5 h of allowed accumulation, the total amount of nickel doubled to 0.02 pg/cell. However, no significant change in the amount of unbound nickel was observed. A similar total accumulation of nickel (0.015 pg/cell) for a 4 h incubation period from 3 X M nickel solution was observed for FM3H cells from C3H mammary tissue (13). These data indicate that relatively high amounts of nickel accumulate in the cells studied. This effect has been noted

previously and attributed to the fact that the cell membrane is negatively charged, enabling it to compete for nickel binding with other ligands. Nickel internalization by the cell is possible by either active transport, most likely through calcium and/or magnesium channels, or passive diffusion of neutral, lipophilic complexes (14). While the exact nature of the nickel-binding sites in the cell is unknown, studies have pointed to the involvement of phosphate groups of both RNA and to a lesser extent DNA along with proteins, phospholipids, amino acids, mononucleotides, and other ligands. These extensive binding capabilities within the cell for nickel account for the relatively small proportion of total nickel concentration left unbound. ACKNOWLEDGMENT We thank Drs. J. R. Fish and K. Kasprzak for helpful comments and suggestions. Registry No. Ni, 7440-02-0; nickel poly(tetrakis(3-methoxy4-hydroxyphenyl)porphyrin),126752-50-9.

LITERATURE CITED Fieischmann, M.; Pons, S.; Roiison, D. R.; Schmidt, P. P. Ukramicroelectrodes; Datatech Systems Inc.: Morganton, NC, 1987. Guadalupe, A. R.; Abruna, H. D. Anal. Chem. 1985, 5 7 , 142-149. Malinski, T.; Ciszewski, A,; Fish, J. R.; Czuchajowski, L. Anal. Chem. 1990, 62, 909-914. Fishbein, L.; Mehiman, M. A. Advances in Modern Environmental Toxicology; CRC Press: Boca Raton, FL, 1987; pp 145-183. Costa, M. Env. Heanh. Perspect. 1989, 8 1 , 73-76. Coogan, T. P.; Latta, D. M.; Snow, E. T.; Costa, M. Crit. Rev. Toxicol. 1989, 19, 341-384. Ponchon, J. L.;Cespugiio, R.; Gonon, F.; Jouvet, M.; Pujol. J. F. Anal. Chem. 1979, 51, 1483-86. Tanaka, K.; KashiwaQi, N. J. Electroanal. Chem. Interfacial Eiectrochem. 1989, 275, 95-98. Turner, A. P. F.; Karube, I.; Wilson, G. S. Biosensors Fundamentals and Applications : Oxford University Press: Oxford, New York, Tokyo, 1987. Malinski, T.; Ciszewski, A,; Bennett, J. E.; Czuchajowski, L. Proceedings of the Symposium on Nickel Hydroxide Nectrodes; The Electrochemical Society Meeting, Hollywood, FL, October 1989; Corrigan. D. A., Zimmerman, A. H. Eds.; The Electrochemical Society: Pennington, NJ. 1990; pp 177-193. Josowicz, M.; Potje, K.; Liess, H.-D.; Besenhard, J. 0.; Kurtze, A. Electrochemistry, Sensors and Analysis; Elsevier: Amsterdam, 1987: pp 87-104. Whiteley, L. D.; Martin, C. R. Anal. Chem. 1987, 59, 1746-1751. Nishimura, M.; Umeda, M. Mutat. Res. 1979, 6 8 , 337-349. Nieboer, E.; Stafford, A. E.; Evans, S. L.; Dolovich, J. Nickel in the Human Environment; Sunderman, F. W., Ed.: IARC Science Publication No. 53; International Agency for Research on Cancer: Lyon, 1984; pp 321-331.

RECEIVED for review June 20, 1990. Accepted October 26, 1990. This work was supported by a grant from William Beaumont Hospital Research Institute.

Tissue Bioelectrode for Eliminating Protein Interferences Joseph Wang,* Li Huey Wu, Sandra Martinez, and Juanita Sanchez Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003 One of the major difficulties in clinical applications of voltammetry stems from the adsorption of proteins onto the electrode surface. A degraded and irreproducible response characterizes these deactivation processes. Such loss of electrode activity is commonly referred to as “fouling” or “poisoning”. Various approaches have been used to alleviate the protein fouling problem. Permselective coatings represent one useful avenue. Size-selective cellulose acetate films have been particularly attractive for this task (1,2). Reactivation

* To whom correspondence should be addressed.

schemes, based on electrochemical or laser pulses, have also proved useful for the prevention of surface passivation effects (3,4).

This paper describes a novel and highly useful approach, based on tissue-modified electrodes, for the prevention of biofouling. The rich biocatalytic activity of tissue-containing surfaces is exploited for in situ enzymatic digestion of interfering proteins. The use of plant or animal tissues in place of isolated enzymes can offer attractive properties for biosensing probes, including extended lifetimes, high enzymatic activity, and low cost ( 5 , 6). These advantages have been

0 199 1 American Chemical Society 0003-2700/91/0363-0398$02.50/0

ANALYTICAL CHEMISTRY, VOL. 63, NO. 4,FEBRUARY 15, 1991

399

illustrated for effective detection of numerous substrates of enzymes present in these cellular materials (7).We have also illustrated recently the utility of tissue electrodes for eliminating interferences from coexisting electroactive constituents (8). For example, zucchini electrodes, rich with the enzyme ascorbic acid oxidase, were employed for effective depletion of ascorbic acid from the surface. Similar enzymatic digestion is employed in the present work for eliminating major interferences from coexisting surface-active proteins. This concept is illustrated by using a papaya-containing carbonpaste electrode. The presence of the protease enzyme papain in the papaya tissue (9) effectively eliminates protein interferences. The broad action of papain toward the cleavage of numerous peptide bonds (10) makes it an ideal choice for this antifouling action. The mixed tissue-carbon-paste configuration ( I I), with its immediate proximity of biocatalytic and sensing sites, is particularly attractive for this task, as it rapidly "destroys" the approaching protein. The smaller peptides resulting from the cleavage of the protein do not passivate the surface. The needs for protective layers or reactivation schemes are thus greatly alleviated. These advantages are illustrated below for measurements of numerous compounds of biological and pharamceutical significance, in the presence of representative proteins. EXPERIMENTAL SECTION Apparatus. Experiments were performed with a Bioanalytical System (BAS) Model VC-2 voltammetric cell that had a working volume of 10 mL. The cell was joined to the working electrode, reference electrode, (Ag/AgCl (3 M NaCl), Model RE-1, BAS) and the platinum-wire auxiliary electrode through holes in its Teflon over. Voltammograms were recorded with an EG&G PAR Model 264A voltammetric analyzer and a Houston Instruments X-Y recorder. The papaya-modified electrode was prepared in the following manner. A 0.2-g section of the papaya (from ca. 2 mm below the skin of the fruit) was ground with a mortar and pestle and hand-mixed (with spatula) with 1.8 g of carbon paste (made of 60% (w/w) graphite powder and 40% (w/w) mineral oil). Mixing proceeded for 20 min to ensure uniform distribution of the tissue. A portion of this modified carbon paste was packed into a 2 mm diameter cavity of a cylindrical Teflon shaft. The surface was smoothed on a weigh paper, and was preconditioned,by immersion for 12 h (at 5 "C) in a 5 mM cysteine solution. Reagents. All solutions were prepared with doubly distilled water. The supporting electrolyte was 0.05 M phosphate buffer (pH 7.4). Acetaminophen, dopamine, uric acid, cysteine, papain (E.C.3.4.22.2, crude type), theophylline, albumin, globulin,gelatin, and casein (Sigma) were used without further purification. The papayas used in this study were purchased from a local grocery store. RESULTS AND DISCUSSION Papain is the main protein constituent of the latex of the fruit, leaves, and trunk of the papaya tree (Carica papaya). Such thiol protease breaks down proteins by hydrolyzing specific peptide bonds. This protein-digesting property has found a wide range of industrial uses (e.g. meat tenderization), but has not been exploited for analytical purposes. Figure 1 illustrates the prevention of protein biofouling achieved by incorporating the papaya tissue within the carbon-paste matrix. I t shows typical differential pulse voltammograms M acetaminophen, in the presence of increasing for 2 X levels of albumin (200-1000 ppm), as obtained a t the unmodified (A) and papaya-modified (B) carbon-paste electrodes. The plain electrode exhibits rapid depressions (up to 63%) and shifts of the acetaminophen peak on successive additions of the proteins. In contrast, essentially the same acetaminophen response is observed at the tissue electrode in the absence and presence of albumin. Apparently, the tissuebased papain rapidly destroys the protein molecules upon their approach to the surface, thus minimizing passivation effects.

POTENTIAL (V) Figure 1. Differential pulse voltammograms at the plain (A) and tissue (10% w/w papaya) (B) electrodes, for 2 X lo-' M acetaminophen: (a)analyte alone; (b-f) same as a but after successive additions of 200

ppm albumin. Conditions: scan rate, 10 mV/s; amplitude, 25 mV; electrolyte, 0.05 M phosphate buffer (pH 7.4).

POTENTIAL (V:

Figure 2. Differential pulse voltammograms,at the unmodified (bottom) M dopamine in the and tissue-modified (top)electrodes, for 5 X absence (a)and presence (b) of 100 ppm casein (A), albumin (B), and gelatin (C). Other conditions are given In Figure 1.

The slightly smaller peak at the tissue electrode is attributed to the reduced graphite content. Significantly larger sensitivity losses characterize the use of permselective protective layers (e.g., ref 2). The interference of different proteins upon the measurement of various oxidizable analytes can be effectively addressed a t the papaya-modified electrode. Figure 2 compares differential pulse voltammograms for dopamine, as obtained before (a) and after (b) addition of 100 ppm casein (A), albumin (B), and gelatin ( C ) . All three proteins result in appreciable (45-90%) loss of the activity of the plain electrode (bottom). The tissue electrode, in contrast, exhibits a highly stable dopamine response, with no apparent change in the peak currents (top, a vs b). Similar advantages were observed M uric acid in the presence of in measurements of 2 X 400 ppm globulin and of 2.5 X M theophylline in the

400

ANALYTICAL CHEMISTRY, VOL. 63, NO. 4, FEBRUARY 15, 1991

I

I

IOOC

7

I/

i X l 0 0

i0

40 C o n c . ,PPm

Conc., p p m

201

0

u 10

I5

PAPAYA, X w

Figure 3. Effect of the paste composition (tissue “loading”)on the diminution of the 1 X M acetaminophen peak in the presence of 200 ppm albumin. Other conditions are given in Figure 1.

presence of 200 ppm casein (not shown). The paste composition has a profound effect on the prevention of protein interference. Figure 3 shows the dependence of the acetaminophen-peak diminution (in the presence of 200 ppm albumin) upon the tissue loading. As expected from the increased protease activity of the electrode, the protein interference decreases upon increasing the amount of tissue in the paste (from 50 to 10% depression at 0-7.5% (w/w) papaya, respectively). A 10% (w/w) tissue loading offers complete elimination of the protein interference. It should be noted, however, that the paste composition needed for effective prevention of protein fouling must be adjusted to suit the conditions of each particular case (mass transport, protein and its level, etc.). No apparent difference in the resistance to biofouling was observed when papaya sections from different locations in the fruit were employed. Tissue electrodes are particularly attractive for the task of prevention of interferences, because cellular materials represent a rich source of biocatalytic activity. Unlike substrate measurements (for which control of the enzymatic activity is often desired), the destruction of interferences requires a large excess of the enzyme, as provided by the surface-containing tissue. Additional activation of the crude papain was obtained by a simple preconditioning step-immersion the tissue electrode in a cysteine solution-aimed at incorporating essential sulfhydryl groups. Other sensing advantages of the tissue-containing papain are its broad optimum pH (6-7.5), heat stability (up to 80 “C), and extremely low cost. No apparent interference from other enzymatic pathways, that may be present in the tissue, or from the variety of smaller peptides resulting from the papain action (IO) was observed throughout this study. Control experiments, utilizing the enzyme (papain)-modified carbon pastes (10% w/w) yielded similar elimination of protein interferences (not shown). This fact, coupled with the cysteine dependence, supports that it is the papain in the papaya which is responsible for the antifouling effect (although other proteases present in smaller levels in the papaya latex, chymopapain and papaya peptidase, may assist in the protection process). The high enzymatic activity, characteristic of tissue bioelectrodes, results in excellent resistance to biofouling over a wide concentration range of different proteins. For example, Figure 4 shows the effects of albumin (A), globulin (B),casein (C), and gelatin (D) on the voltammetric response of acetaminophen. Successive additions of these proteins, over the 200-1000 ppm range, caused significant peak current depressions a t the unmodified electrode (b). With the papaya-modified electrode, in contrast, the quantitation of the drug is not affected by the presence of the proteins over their entire concentration range (a). In order to be practical for routine biosensing applications, the resistance to fouling should be

conc . p p m

Figure 4. Effect of albumin (A), globulin (B), casein (C), and gelatin (D) on the voltammetric peak of 2 X lo-‘ M acetaminophen,at the tissue (a)and plain (b) carbon-paste electrodes. Other conditions are given in Figure 1.

maintained over a long period of time. The stability of the response of the tissue electrode was tested by checking its behavior, in the presence of 200 ppm globulin, during a long run of 24 successive determinations of dopamine, carried out over a 2-h period. No apparent change in the sensitivity was observed throughout this operation. Indeed, tissue-containing surfaces have been employed over an entire week, performing hundreds of measurements with excellent prevention of protein passivation effects. In conclusion, the present study illustrates that tissue bioelectrodes can be employed to circumvent protein passivation effects, hence imparting high stability during electrochemical measurements. Such in situ destruction of potential interferences should simplify sample cleanup procedures and eliminates the need for protective membranes or reactivation schemes. Hence, this concept should be highly suitable for routine sensing applications based on finite-current measurements. Other tissues, e.g. pineapple or fig, rich with the proteases bromelain and ficin, respectively, should yield similar advantages. The mixed carbon-paste configuration permits simultaneous incorporation of other biological and chemical modifiers, as well as convenient miniaturization. For example, simultaneous elimination of major electroactive constituents may be accomplished via coimmobilization of enzymes, such as uricase, ascorbic acid oxidase, or tyrosinase, while lipid interferences may be addressed in the presence of lipase. Registry No. Papain, 9001-73-4;carbon, 7440-44-0. LITERATURE CITED (1) Sittampalam, G.; Wilson, G. S . Anal. Chem. 1983, 55, 1608. Wang, J.; Hutchins, L. D. Anal. Chem. 1985, 5 7 , 1536. Room, M.; McCreery. R. L. Anal. Chem. 1988, 58, 2745.

(2) (3) (4) (5) (6) (7) (8) (9)

Wang, J.; Lin. M. S. Anal. Chem. 1988, 6 0 , 499. Rechnitz, G. A. Science (Washington, D.C.)1981, 2 1 4 , 287. Arnold, M. A . Am. Lab. (Fairfield, C T ) 1983, 15(6),34. Wang, J. ,Electroanalysis. in press. Wang, J.; Naser. N.; Ozsoz, M. Anal. Chim. Acta 1990, 234, 315. Liener, I.E. The Sulfhydryl Porteases in Food Related Enzymes; Whitaker, J., Ed.; American Chemical Society: Washington, DC, 1974. (IO) Hill, R. I n Hydro/ysis offroteins; Anfinsen. C. E., Anson, M. L., Edsall, J. T.. Richards, F. M., Eds.; Advances in Protein Chemistry; Academic Press: New York, 1965; Vol. 20. (11) Wang, J.; Lin, M. S. Anal. Chem. 1988, 6 0 , 1545.

RECEIVED for review August 6, 1990. Accepted November 7, 1990. This work was supported by grants from the National Institutes of Health (GM 30913-06, GM 07667-14, and RR 08136-16).