790
Anal. Chem. 1904, 56,798-801
LITERATURE CITED (1) Kaiser, R. E. J. Chromatogr. Scl. 1974, 12, 36. (2) Krost, K. J.; Pellizzari, E. D.; Walburn, S.G.; Hubbard, S. A. Anal. Chem. 1982, 54, 810. (3) Brown, R. H.; Purneil, C. J. J. Chromatogr. 1979, 778, 79. I Clark, A. I.; McIntyre, A. E.; Lester, N. J.; Perry, R. J. Chromatrogr. 1982, 252, 147. ' Grimsrud, E. P.; Rasmussen, R. A. Afmos. Environ. 1975, 9 , 1010. Tvson. , - - El. J. - Anal. Letf. 1975. 8 . 807. Rasmussen, R. A.;Hukon, R. S.Bbscience 1972, 2 2 , 294. Tvson. B. J.: Carle. G. C. Anal. Chem. 1974. 46. 610. Pebler, A,;-Hickman, W. M. Anai Chem.-l973, 4 5 315. Seila, R. L.; Lonneman, W. A,; Meeks, S. A. J. Envlron. Sci. Health, PartA 1976. A l l . 121. Lonneman, W.A.;'Bufaiini, J. J.; Kuntz, R. L.; Meeks, S. A. Envlron. Sci. Techno/. 1981, 75, 99. Singh, H. B.: Salas, L. J.; Shigiishi, H.; Scribner, E. Science 1979, 203, 903. Fisher, R. L.; Reiser, R. W.; Lasoski, 8. A. Anal. Chem. 1977, 4 9 , 1821. Fine, D. H.; Rounbehler, D. P.; Sawicki, E.; Krost, K. Envlron. sei. rechnol. 1977, 11, 577.
.
~
----.-.
(15) Lovejoy, D. J.; Vosper, A. J. J. Chem. SOC. 1968, 2325. (16) Sievers, R. National Symposium on Monitoring Hazardous Organic Pollutants in Air, Raleigh, NC, 1980. (17) Bunch, E,; E, D, J , chromatogr, Ig7g, 186, 8 1 , , (18) Rounbehier, D. P.; Reisch, J. W.; Coombs, J. R.; Fine, D. H. Anal. Chem. 1880, 5 2 , 273. (19) Berkley, R. E.; Pellizzari, E. D. Anal. Lett. 1978, 4 , 327. (20) Box, G. E. P.; Hunter, W. G.; Hunter, J. J. "Statistics for Experimenters"; Wiiey: New York, 1978; p 306. J,
RECEIVED for review June 6,1983. Accepted January 16,1984. Although the research described in this article has been funded wholly or in part by the U.S. Environmental Protection Agency through Contract No. 68-02-3423 and 68-02-2998 to the Research Triangle Institute, it has not been subjected to the Agency's required peer and administrative therefore, does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.
Detection of Microbial Cells by Cyclic Voltammetry Tadashi Matsunaga* and Yoichi Namba Department of Applied Chemistry for Resources, Tokyo University of Agriculture & Technology, Koganei, Tokyo 184, J a p a n
A novel method for detecting microbial cells has been developed by applying cycllc voltammetry to a cell suspension of S. cerevislae. A basal plane pyrolytic graphite electrode was employed as a working electrode. An anodic peak current appeared at 0.74 V vs. SSCE. A linear relationship was obtalned between the peak current and the cell concentration In the range (0.1-1.9) X 10' cells.mL-'. The peak currents were reproducible with an average relatlve error of 4 % . The peak currents per 10' cells were almost constant for cells harvested at an exponential growth period and a stational growth period. The peak current was not obtalned from protoplasts of S. cerevlslae. I t was shown that CoA existlng In the cell wall medlated an electron transfer between the cells and the graphite electrode.
Detection of living microbial cells is important in clinical, environmental, and bioindustrial fields. Methods based on colony formation have been used for the detection of viable cells in microbiology (1). However, these methods are time consuming and demand complicated procedures. Recently, various electrochemical methods have been developed for determining viable cell numbers (2-5). The impedance measurement of culture media has been proposed as a method for cell number determination (2,3). A s nutrients are converted to various charged metabolites such as organic acids and other compounds by bacteria, the impedance of media increased with increasing cultivation time. Increase of the impedance is correlated with the initial cell numbers of bacteria in the culture media. This method, however, requires a long incubation time. Wilkins et al. developed an electrochemical method based on the detection of hydrogen molecules produced by bacteria (4). The principle of hydrogen detection is based on the observation that hydrogen ions and molecular hydrogen establish an equilibrium with platinum. This method is applicable to the estimation of cell numbers of Enterobacteriaceae, etc. However, the cell numbers of the
other species of microorganisms cannot be determined by this method. One of the present authors has developed an electrode system composed of a platinum anode and a silver peroxide cathode ( 5 , 6 ) . Cell populations of Saccharomyces cerevisiae and Lactobacillus fermentum were determined by use of this electrode system. Determination of cell numbers of Bacillus subtilis was also carried out by use of an electrode system constructed from two platinum electrodes and a saturated calomel electrode (7). However, the mechanism of current generation was unknown. Furthermore, both systems require a reference system of which a platinum anode was covered with cellulose dialysis membrane because a background current was obtained from a working electrode system. In this paper, a novel method for detecting microbial cells has been developed by applying cyclic voltammetry to a microbial suspension of S. cerevisiae. A basal plane pyrolytic graphite electrode was employed as a working electrode. This study also investigated the mechanism of electron transfer between S. cerevisiae and a graphite electrode. Major efforts are on the identification of an electroactive substance on microbial cells.
EXPERIMENTAL SECTION Materials. Phosphotransacetylase (EC 2.3.1.8) was purchased from P. L. Biochemicals, Inc. (Milwaukee, WI). Coenzyme A was obtained from Sigma Chemical Co. (S. Louis, MO) and acetyl phosphate from Boehringer Mannheim (Mannheim, West Germany). Yeast extract was purchased from Difco Laboratories (Detroit,MI) and Polypeptone from Kyokuto Pharmaceutical Co., Ltd. (Tokyo,Japan). Other reagents were commercially available analytical reagents or laboratory grade materials and were used as received. Deionized water was used in all procedures. Microbial Cells. S. cereuisiae was cultured aerobically at 30 "C for 12 h in 100 mL of a medium (pH 7.0) containing 4 g of glucose, 1 g of Polypeptone, 0.5 g of KH2P0,, and 0.2 g of MgSO,*7H20. Apparatus. Cyclic voltammograms were obtained with a potentiostat (Hokuto Denko, Model HA301), a function generator (Hokuto Denko, Model HB104) and a X-Y recorder (Riken Denshi, F35). Cyclic voltammetry was run on basal plane pyrolytic graphite electrode with a surface area of 0.17 cm2. Unless oth-
0 1984 American Chemical Society 0003-2700/84/0356-0798$01.50/0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984 1,o
t
799
7 1
0
.e c)
E
0,5 -
a 0 Y
E
1
LL
2
0
0,5 E(V) YS
Figure 1. Cyclic voltammograms of a whole cell suspension of S. cerevisiae. The cell concentrations were (a) 2.8 X 10' cells-mL-', (b) 1.9 X 10' cells-mL-I, and (c) buffer solution only. Scan rate was 10 mV.s-l. The experiments were performed at pH 7.0 and ambient temperature (25 f 2 "C).
erwise specified, the electrode was polished with an aqueous suspension of 0.3 wm alumina on a polishing cloth before each run to minimize the undesirable electrochemical signals which arise from surface-absorbed species. The alumina particles were removed from the surface of the electrode via ultrasonic cleaning. The cyclic voltammetry cell was of all-glass construction, approximately 25 mL in volume, incorporating a conventional three-electrode system. The counterelectrodewas a platinum wire and the reference electrode was a sodium chloride saturated calomel electrode. The reference electrode was separated from the main cell compartment by immersion in a glass tube terminated by sintered glass frit. Procedure. The cultured cells were centrifuged at 5 "C and SOOOg, and washed twice with 0.1 M phosphate buffer (pH 7.0). An appropriated amount of cells was suspended in 10 mL of 0.1 M phosphate buffer (pH 7.0), and the cell suspension was continuously bubbled with air. Then, cyclic voltammograms were obtained at ambient temperature, 25 f 2 "C. Determination of Cell Numbers. The number of S. cereuisiae was determined by plating suitably diluted samples of a culture and counting colonies that appeared after 36 h of incubation at 30 " C . Preparation of Protoplasts. Protoplasts of S. cereuisiae were prepared by Zymolyase-5000 (Kirin Brewery Co., Ltd., Tokyo, Japan) treatment. The cells were suspended in 10 mL of hypertonic phosphate buffer solution (pH 7.5) containing 0.6 M KCl. Then, Zymolyase-5000 and 2-mercaptoethanol(O.3%)were added to the cell suspension. The cells were incubated at 30 "C with shaking (30 strokes/min), centrifuged at 5 "C and 8000g,washed with the hypertonic buffer, and resuspended in the hypertonic buffer. Determination of CoA. GOA was determined by the phosphotransacetylase method of Stadtman et al. (8).
RESULTS AND DISCUSSION Cyclic Voltammetry of Microbial Cells. Figure 1 shows the cyclic voltammograms of a whole cell suspension of S. cereuisiae, obtained a t pH 7.0 a t the pyrolytic graphite electrode in the potential range of 0 to 1.0 V vs. SSCE. An anodic wave appeared at 0.74 V vs. SSCE on the first scan in the positive direction. Upon scan reversal, no corresponding reduction peak was obtained. This shows that the electrode reaction of microbial cells is irreversible. The oxidation peak current was found to increase linearly with the square root of the scan rate as expected for a totally irreversible reaction. Figure 2 shows the effect of p H on peak potentials. The slope of peak potentials vs. pH line was 60 mV above pH 5.5. There was a distortion of this linearity at pH 5.5. These results indicate that two proton are transferred above pH 5.5. The p H of sample solution was adjusted to 7.0 for microbial detection.
a
PH
1,o
SSCE
6
4
I
1
Figure 2. Peak potentials as a function of pH. The cell concentration was 2.6 X lo9 cells.mL-'. The experiments were performed at pH 7.0 and ambient temperature (25 f 2 "C). pH of the cell suspension
was adjusted with
0.1
N HCI and 0.1 N NaOH solutions.
Table I. Peak Current of Cells Harvested at Various Incubation Times
incubation time, h
cell population peak current/ in culture medium, cells, l o 8 cells (PA/ IO8 cells.mL-I) mL-
6 8
4.0 x 107 1.7 x l o 8
10
2.8 X 10' 3.0 x l o 8
12 14 16
18 20 22 24
3.3 x 3.5 x
lo8 lo8
4.0 x l o 8 3.8 x l o 8
3.8
X
3.8 x
lo8 lo8
0.64 0.67 0.31 0.31
0.30
0.31 0.30 0.30 0.08 0.03
Determination of Cell Concentration. The relationship between the peak currents and the cell concentration of S. cereuisiae was studied. A linear relationship was obtained between the peak current and the cell concentration in the range over (0.1-1.9) x lo8 cells-mL-l. The peak current was reproducible with an average relative error of 4% when a cell suspension (0.5 X lo8 cells-rnl-l) obtained from the same culture broth was employed for experiments. These results show that cell numbers of S. cereuisiae can be determined from the peak current of cyclic voltammetry. Table I indicates the peak currents of cells incubated for 6-24 h. The peak currents per lo8 cells were relatively high a t 6-8 h (an early exponential growth period). The peak currents obtained from cell suspension were almost constant at 10-20 h (an exponential growth period and a stational period). However, a little deviation was observed in exponential growth. This may be caused by budding of the yeast. Many buds were observed at this time with a light microscope. However, the peak currents were very low for old cells incubated more than 22 h. The peak current seems to be closely related with viability and metabolism of microbial cells. Mechanism of C u r r e n t Generation. Electron transfer from microbial cells to graphite electrode is expected to be closely correlated with formation and generation of coenzymes. Therefore, the peak current seems to be related with metabolic pathway. Then, metabolic inhibitors such as rotenone, antimycine, cyanide, and arsenite were added to cell suspension of S. cereuisiae. Rotenone, antimycine, and cyanide inhibit the mitochondrial electron-transport chain. Although rotenone specifically inhibits electron transfer within NADH dehydrogenase, antimycine inhibits electron flow between cytochromes b and c, and cyanide blocks electron flow between the cytochrome oxidase complex and 02,the addition of ro-
800
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
15
-
Q =L
10
+S 0 3
5
1,o
0,s
0
0
E N ) 41s SSCE Cyclic voltammograms of protoplast suspension of S . cerevisiae. Scan rate was 10 mV.s-'. The concentrations of (a) protoplasts and (b) whole cells were 5 X 10' cells-mL-', respectively. The experiments were performed at pH 7.0 and ambient temperature (25 f 2 "C). Flgure 3.
0
5
1(1
15
20
Time (rnln) Flgure 4. Time-course of peak currents obtained from (0)eluent and (0)whole cells when whole cells of S.cerevlshe were sonicated. The cell Concentration was 9 X 10' cells.mL-'. After whole cells were
sonicated in the phosphate buffer solution (pH 7.0), cells were centrifuged at 5 OC and 80009 and the eluent was obtained. Collected cells were resuspended in the buffer solution. Cyclic voltammograms were obtained for eluents and cell suspension.
tenone (7.6 mM), antimycine (5.7 mM), and cyanide (10.8 mM) did not decrease the peak current of cyclic voltammogram of cell suspension (2.4 X lo8 cells-mL-'). These results suggest the peak current generation is not correlated with the oxidative phosphorylation which occurs inside mitochondria. Arsenite is known to inhibit pyruvate dehydrogenase. The peak current of cyclic voltammogram decreased from 4.8 PA to 3.7 pA when 10 mM of arsenite was added to the cell suspension (2.4 X lo8 cells-mL-'). Therefore, the generation of peak current is correlated with pyruvate dehydrogenase and the citric acid cycle. Figure 3 shows the cyclic voltammograms of protoplasts and whole cell suspensions. The peak current was not obtained from protoplasts of S. cerevisiae. This experimental result indicates that electroactive substances such as coenzymes bind to the cell wall. Then, cell-wall-bound compounds were eluted by sonicating the whole cells in the buffer solution. Figure 4 shows the time-course of peak currents obtained from eluent and whole cells. The peak current from whole cells decreased. On the other hand, the peak current of the eluent which appeared a t 0.65 V vs. SSCE increased gradually. Since sonication did not affect the number of viable cells in the buffer solution, these results indicate that electroactive substances
3-5
0 E (V) Flgure 5. Cyclic voltammograms sonicated cells, and (c) whole cells. The cell concentration was 9 X 10' pared as described in Figure 4. The
1,o SSCE
of (a) CoA, (b) the eluent from CoA concentration was 3.6 mM. cellsemL-'. The eluent was preeluent contained 3.6 mM of CoA.
in the cell wall eluted in the buffer solution with sonication and were detected electrochemically. The absorption of the eluent at 260 nm, which is corresponding to the adenine ring, also increased with increasing the peak current of the eluent when the cell suspension was sonicated. Cofactors such as NADH, NADPH, FMNHZ, and CoA which have the adenine ring can be oxidized electrochemically. However, the absorption at 340 nm corresponding to NADH and NADPH and that a t 445-450 nm corresponding to FMNHz were not obtained from the eluent. The experimental half-wave potential of NADH and NADPH has ranged from 0.35 V to 0.75 V vs. SCE a t carbon electrodes (9-11). FMNHz and FADHz were reported to be oxidized electrochemically a t -0.4 V vs. SCE (12). The cyclic voltammograms of CoA, the eluent, and whole cells are shown in Figure 5 . The peak currents of CoA was observed at 0.65 V vs. SSCE. The peak potential of CoA is similar to that of the eluent from sonicated cells. On the other hand, the peak current was observed a t 0.35 V vs. SSCE for NADH at the BPG electrode. Therefore, CoA in the eluent from the sonicated cells was determined enzymatically by the method of Stadtman et al. (8). As a result, 3.6 mM of CoA was detected in the final eluent. The concentration of CoA in the final eluent was also calculated from the peak current because a linear relationship was obtained between the CoA concentration and the peak current. The CoA concentration electrochemically determined was 3.9 mM which is close to the value enzymatically determined. In order to exclude the possibility that another compound may be causing current, CoA in the eluent was enzymatically converted to acetyl CoA with acetyl phosphate by phosphotransacetylase. As a result, the peak current of the eluent decreased from 5.4 PA to 0.5 @A. This indicates that CoA mainly contributes to the peak current of the eluent. The concentration of CoA also increased with increasing the peak current of the eluent when microbial cells were sonicated. Therefore, increase of peak current obtained from the eluent was attributed to increase of CoA concentration in the buffer solution. On the other hand, as CoA existing in the cell wall was eluted in the buffer solution by sonicating the whole cell, the peak current obtained from the whole cells decreased. These results indicate that CoA existing in the cell wall mediated an electron transfer between cell and graphite electrode. Then, CoA existing in the cell wall of the old cells harvested at 24 h was also eluted by sonicating and determined enzymatically. As a result, only 1.8 mM of CoA was detected. Therefore, the decrease of the
Anal. Chem. 1984, 56,801-806
peak current obtained from the old cells is attributed to the decrease of CoA content of the cell wall. As shown in Figure 5, the peak potential of the eluent, which is similar to that of CoA, was more positive than that of the whole cells. The transport characteristic of CoA to the electrode surface is estimated from Figure 4 for the two cases of free and cellbound CoA. The increasing slope of current obtained from the free CoA with time is about 5 times the decreasing slope of the current from the bound CoA. This result shows the diffusion of CoA being present in the cell wall is lower than that of CoA in solution. Therefore, the peak potential of whole cells is higher than that of CoA solution and the eluent from sonicated cells, and the peak current obtained from whole cells is also lower than that of CoA solution and the eluent. The microbial detection method described here differs in principle from the previous electrode system (5-7). Because the platinum anode of the previous electrode systems was controlled at 0.20-0.35 V vs. SSCE and the peak current of this system was obtained at 0.74 V vs. SSCE. Oxidation of CoA does not occur at 0.20-0.35 V vs. SSCE. Moreover, the current from the previous system was affected by the concentration of dissolved oxygen, whereas the oxygen did not affect the peak current and potential in this system. The novel concept described here for determining cell number of S. cereuisiae lays the ground work for the devel-
80 I
opment of methods detecting microbial strains. Further developmental studies in our laboratory are now directed toward determining and recognizing various species of microorganisms. Registry No. CoA, 85-61-0.
LITERATURE CITED Postage, J. R. "Methods in Microbiology"; Norris, J. R., Ribbons, D. W., Eds.; Academic Press: New York, 1969; Vol. 1, pp 611-621. Hadiey, W. K.; Senyk, G. "Microbiology-1975"; Schlesslnger, D.,Ed., American Society for Microblology: Washington, DC, 1975; pp 12-21, Zafari, Y.; Martin, W. J. J. Clln. Microbiol. 1977, 5 , 545-547. Wilkins, J. R.; Young, R. N.; Boykin, E. H. Appl. Environ. Microbiol. 1977, 35, 214-215. Matsunaga, T.; Karube, I.; Suzuki, S. Anal. Chim. Acta 1978, 98, 25-30. Matsunaga, T.; Karube, I.; Suzuki, S.Appl. Mviron. Microbiol. 1970, 37, 117-121, Matsunaga, T.; Karube, I.; Suzuki, S. Eur. J . Appl. Microblol. Biotechno/. 1980, 70, 125-132. Stadtman, E. R.; Novelll, G. D.; Lipman, F. J. Biol. Chem. 1951, 797, 365-376. Leduc, P.; Thevenot, D. H. Bioelectrochem. Bioenerg. 1974, 7 , 96-107. Blaedei, W. J.; Henklns, R. A. Anal. Chem. 1974, 46, 1952-1955. Blaedel, W. J.; Jenkins, R. A. Anal. Chern. 1975, 47, 1337-1343. Dryhurst, G., Ed. "Electrochemistry of Biological Molecules"; Academic Press: New York, 1977; pp 365-389.
RECEIVED for review September 9,1983. Accepted January 11, 1984.
Potentiometric Digoxin Antibody Measurements with Antigen-Ionophore Based Membrane Electrodes M. Y. Keating and G . A. Rechnitz*
Department of Chemistry, University of Delaware, Newark, Delaware 19716
We describe and Illustrate a technlque, potentlometrlc ionophore-modulation Immunoassay (PIMIA), for the measurement of antibodies with conlugate based membrane eiectrodes. Fundamental operatlng varlabies for the technique are examined and demonstrated for the case of antibodies to the cardlac drug dlgoxln. Detection ilmits In the pg/mL range, with high seiectivlty over other antibodies and proteins, are readlly attalned. Through a competttlve blnding approach, the selective measurement of digoxln itself is also shown to be possible with this technique.
Antigen and antibody measurements are of importance in clinical chemistry, physiology, and modern biotechnology. Considerable effort has been made to develop potentiometric membrane electrode sensors for this purpose, but contrary to the more straightforward enzyme-immunoassay techniques where a potentiometrically measurable product is liberated (1-4),it is much more difficult to couple antibody-antigen reactions to membrane electrodes. In this paper we attempt to lay the foundation for a class of potentiometric membrane electrodes which respond to specific antibodies through modulation of a background potential fixed by a marker ion and to illustrate this concept with the development of a selective electrode for antibodies to digoxin, a steroidal cardiac drug. For lack of an established descriptive term, we call this technique potentiometric ion0003-2700/84/0356-0801$01,50/0
ophore-modulation immunoassay (PIMIA). The principle of the method is simple. An antigen or hapten corresponding to the antibody to be measured is chemically coupled to an ionophore to form an antigen-carrier conjugate. The conjugate is incorporated into a plastic support membrane and that membrane is mounted in the sensing tip of a conventional potentiometric membrane electrode (Figure 1). The resulting electrode is exposed to a constant activity of a marker ion chosen for its compatibility with the ionophore portion of the conjugate, under conditions which produce a stable and reproducible background potential. When an antibody capable of binding the antigen portion of the conjugate is added to the background electrolyte, a potential change (hE) proportional to the antibody concentration is produced. It will be shown below that other antibodies or nonspecific proteins cause negligible interference with the determination of the primary antibody. In the present study, the selectivity and high affinity of digoxin antibodies raised in rabbits for the drug are especially favorable since the intrinsic affinity constant has been reported to be 1.7 X 1O1OpUl-l(5). Moreover, the ionophores benzo-15-crown-5 and cis-dibenzo-18-crown-6 have excellent solubility in the poly(viny1 chloride) support membranes employed. Although we have previously reported on some of the construction details (6) and preliminary analytical limits (7) of earlier electrodes, the present paper, detailing a full investigation of the newly studied digoxin antibody electrode system, represents the first comprehensive effort to interpret 0 1984 American Chemical Society
