Anal. Chem. 1989, 61,2388-2391
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still incompletely unraveled but should most certainly be explained by the concentration dependence of the relationships associating an apparent velocity to a concentration (7). Under the experimental conditions leading to a reversal of the direction of the band asymmetry, the velocity associated to a concentration decreases with increasing concentration, at the opposite of what happens in the case of the classical Langmuir isotherm. Then the position of the concentration shock on the profile shifts from the front to the rear and most phenomena due to the competition between the components of a mixture are reversed (8). Some consequences of the new retainment and pull back effects have no doubt been observed previously by separation chemists trying to perform preparative chromatography with normal-phasesystems and using strong solvents. These effects have not been mentioned yet in the literature, to the best of our knowledge. They are impossible to understand if the major role of the third component of the competitive interaction process, the strong solvent or organic modifier, is not recognized. The use of detectors that are selective against the components of the mobile phase, while quite reasonable in principle, contributes to obfuscate the issue by hiding the system peaks, the presence of which could alert the observer. Comparison between the signals given by UV and refractive index detectors should be a natural reflex of chromatographers
when an overloaded column gives unexpected signals.
ACKNOWLEDGMENT We are grateful to Hewlett-Packardfor the gift of the Model 1090A Liquid Chromatograph and Datastation.
LITERATURE CITED (1) Golshan-Shlrazi, S.; Guiochon, G. J . Chromatcgr. 1989, 461,1. (2) Golshan-Shirazl, S.;Gulochon, G. J. C h m t o g r . 1989. 467, 19. (3) Golshan-Shirazi, S.;Guiochon, G. Anal. Chem., preceding paper in
this Issue. (4) Golshan-Shlrazl, S.; Ouiochon, G. J . Fhys. Chem. 1989, 93, 4143. (5) Ghodbane. S.; Guiochon, G. J. Chromatcgr. 1988, 440, 9. (8) Oulochon, G.; Gdshan-Shlrazi, S.; Jaulmes, A. Anal. Chem. 1988, 60, 1858. (7) Lin, B.; Golshan-Shirazl, S.; Ma, Z.; Gulochon, G. And. Chem. 1988, 60, 2847. (8) Goishan-Shirazi, S.; Guiochon, G., unpublished data, 1989.
RECEIVED for review May 22,1989. Accepted August 4,1989. This work was supported in part by grant CHE-8901382 from the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory.
Microbial Sensor for Preliminary Screening of Mutagens Utilizing a Phage Induction Test Isao Karube* and Koji Sode Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153, Japan
Masayasu Suzuki and Takashi Nakahara Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, J a p a n
For the prellmlnary screenlng of mutagens, a novel microbial sensor system was developed utlllzlng a phage Induction test. Escherlchla coU lysogenlc straln GY5027 and nonlysogenlc straln GY5026 were used In thls study. The number of llvhg cells was determined by measurlng the resplratlon of cells lmmoblllzed onto an oxygen electrode. The lnjectlon of a mutagen, such as AF-2 and MNNG, caused the phage Induction In the lysogenlc straln, resulting In the decreased resplration of only the lysogenk straln I"obl#red onto the oxygen electrode but not of nonlysogenlc straln. The rate of current Increase correlated well wlth the concentration of mutagens. The sensor responses to the antlMotlcs and bacterkldes were deflnltely dlfferent from those of mutagens. Therefore, utllizatlon of thls mlcroblal sensor system makes posslble the estlmatlon of a substrate's mutagenicity.
INTRODUCTION The mechanisms of carcinogenesis are currently being investigated by many researchers. It is well-known that one of
* To whom correspondence should be addressed.
the most contributing factors for human cancer are environmental carcinogenic substances. Knowledge of carcinogens is a way to prevent human cancer. However, many new chemicals have been investigated every year, and it is almost impossible to estimate carcinogenicity of all chemicals, since conventional tests for the estimation of carcinogenicityrequire complicated and time-consuming procedures. Besides, the carcinogenicity of the substrate is now considered to have a good correlation with the mutagenic activity. Thus, with the advent of preliminary screening of carcinogens, numerous mutagenic tests have been developed. The application of microorganisms, Bacillus subtilis Rec- (rec-assay) ( I ) , Salmonella typhimurium hysthidine auxotrophe (Ames test) (2, 31, and Escherichia coli lysogenic strain (induction test) (4), to mutagenic tests is widely accepted as is a relatively simple yet reliable screening method. Luminous bacteria were also utilized to detect mutagenic compounds (5,6). However, these methods require a long period of microbial incubation. Besides, several enzyme and microbial sensors have been developed combining immobilized biocatalist and electrochemical transducers to simplify several biochemical based analyses (7). Our group has already developed two types of microbial sensors for the preliminary screening of mutagens. These sensors are based on either the Ames test (8) or the
0003-2700/89/0381-2388$01.50/0 C 1989 American Chemical Society
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Z 30
-
7
P
Y
20
U a, u c
10 0 3
2
1
0
1
2
Cell number X lo-* Figure 2. Relationship between current decrease and cell number of E . coliGY 5026 and GY 5027: ( 0 ) GY 5026; (0)GY 5027.
Flgure 1. Schematic diagram of microbial sensor system for the screening of mutagens: (1) membrane filter immobilizing E . coli GY 5026, nonlysogenic strain; (2) membrane filter immobilizing E . coliGY 5027, lysogenic strain; (3) Teflon gas permeable membrane; (4) GY 5026 electrode; (5) GY 5027 electrode; (6) Pt cathode; (7) Pb anode; (8)
recorder.
rec-assay (9). However, the sensor that utilizes the Ames test requires 6 h of incubation for the assay. The mechanisms of rec-assay involve unknown biological features. Moreau et al. have reported on the estimation of mutagenicity by measuring the rate of phage induction within 1 day (IO). This measurement was based upon the phage induction rate of Escherichia coli lysogenic strain. The E. coli lysogenic strain has its phage DNA integrated with in the chromosome (prophage). If the strain is treated with chemical compounds which cause damage to the DNA, a RecA protein (protein responsible for DNA repair) is activated, which subsequently decomposes the repressor protein of phage. As a result, the phage is expressed in the cell. Consequently E. coli is lysed and thereby manifested by the formation of a plaque. Moreau et al. has utilized this principle by incubating an E. coli lysogenic strain in the presence of a mutagen. The number of resulting plaques were then measured as an indicator of mutagenicity. Ikeda et al. have also reported the estimation of mutagenicity by measuring @-galactosidaseactivity released by phage induction (11). This test took only 5-6 h. The induction occurred rapidly in the presence of the mutagen with the mechanism being studied in detail. Therefore, by combination of the induction test with the principle of microbial sensor, a rapid and simple method for estimating mutagenicity is expected. In this study, the preliminary screening of a mutagen is described by implementing a phage induction test and a microbial sensor.
with slight suction. The membrane thus prepared was then affiied to a Teflon membrane of a Clark-type oxygen electrode (Type A, Able Co., Tokyo Japan), and covered with dialysis membrane. The resulting microbial sensor system is shown in Figure 1. The sensor system consisted of two electrodes comprised of either the GY5026 or the GY5027 microbial strains. Assay Procedure. AF-2,4NQO, and MNNG were dissolved. in dimethyl sulfoxide. Mytomicin C was dissolved in distilled water. Each solution was diluted with 0.1 M phosphate buffer to obtain a pH 7.0. Varying concentrations of each sample were also prepared. The microbial sensor system was immened in a 50-mL reaction vessel containing 30 mL of medium (peptone0.8%, trypton 0.5%, NaClO.5%, pH 7.0, vessel and medium were sterilized for 20 min at 120 O C , 1.2 atm). After the output current reached a steady state, the mutagen preparation was injected onto the reaction vessel. The electrode currents were displayed on a recorder. The mutagenicity of the sample was estimated by the increase in the output current of the microbialsensor containing the immobilized lysogenic strain and reflected the decreased number of viable cells caused by phage induction.
RESULTS AND DISCUSSION Measurement of a Number of Viable Cells Immobilized onto the Electrode. The correlation between the cell number and the output current from the electrode was measured by changing the number of cells immobilized onto the porous acetylcellulose membrane. When the microbial electrode was immersed in the solution, the respiration of microorganisms increased. Subsequently, current output of the oxygen electrode decreased. Figure 2 shows the correlation between current decrease in steady state and cell number immobilized onto the electrode. A good linear correlation was observed between the current decrease and the cell number between the range of (0.5-2.0) X lo8 cells. In this range the EXPERIMENTAL SECTION number of viable cells was determined by measuring the Chemicals. Mitomycin C (Sigma Co.), 4-nitroquinoline N current output from the oxygen electrode. In addition, no oxide (4NQO) (Tokyo Kasei Co.), N-methyl-N'-nitro-N-nitrosoobvious difference was observed between the slope of figure guanidine (MNNG) (sigma), and 2-(2-furyl)-3-(5-nitro-2-furyl)- for GY5026 and GY5027. acrylamide (AF-2) (provided by Professor Tsuneo Kada, DeFor the estimation of substrate mutagenicity, the number partment of Induced Mutation, National Institute of Genetics, of viable cells will be used as an indicator. Therefore, the Shizuoka, Japan) were used in this study. All other reagents were change of the number of immobilized cells should be detected of analytical grade. even if its number increases. Thus, in the succeeding exMicroorganisms. Escherichia coli, lysogenic strain (GY5027) periments, the number of cells immobilized onto the electrode and nonlysogenic strain (GY50261, were provided by Professor R. Devoret (Section de Radiobiologie Cellulaire, Laboratoire was set to 1.6 X lo8 cells. d'Enzymologie, C.N.R.S., 91190 Gif-sur-Yvette, France). Both Time Course of Sensor Response to AF-2. Figure 3 strains were cultivated in a yeast medium composed of yeast shows the time course of the sensor response after AF-2 adextract 0.5%, trypton 1%,NaCl 1%,glucose 0.2%, &SO4 0.04%, dition in the reaction medium (AF-2 concentration, 0.2 pg citric acid 0.04%, K2HP04,0.25%, and NaNH4HPO4.4H20 0.07% mL-l). After 2 h of incubation, the output current from the (w/v). After 2 h of incubation at 37 "C in the yeast medium, cells electrode-immobilized GY5027 (lysogenic strain) gradually were centrifuged and utilized for the microbial sensor. increased, while no current change was observed even after Construction of Microbial Sensor System. A 4-mL portion 4 h of incubation from the electrode immobilized GY5026 of either cell suspension GY5026 or GY5027 (4 X 10' cells mL-') (nonlysogenic strain). The current increase observed in was dropped onto a porous acetylcellulose membrane (Millipore GY5027 was reflected by the decrease in the number of living Co. type HA, 0.45 pm pore size, 25 mm diameter, 150 pm thick)
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/
Table I. Sensor Responses to the Various Mutagens and the Other Chemicals
rate of current increase, ~ A l h concn, pg/mL
GY5027
electrode
GY5026 electrode
Mutagen 4NQ0 I
I
1
2
J
0.4 1.0
3.0 0.3 0.8 1.5
mitomycin C
3
Time ( h J
Figure 3. Sensor responses to AF2 (0.2 ccg/mL) addition: (---) re-
MNNG
10
20 30
sponse of GY 5026 electrode; (-); response of GY 5027 electrode. The measurements were attempted at 37 OC,pH 7.0, and with 1.6 X l o 8 cells.
2.5 8.0 9.5 4.5 7.5 13 2.0 2.5 5.0
0 1.0 0 1.0 2.0 0 0 1.0
3.0 16 60 0 3.0 7.0 1.5 4.0 6.0
4.0 18 70 0 4.0 6.0 1.0 3.5 7.5
0
Other Chemicals
benzalkonium chloride kanamycin
neomycin
10 20 40 10 20 40 10 20 40
B. subtillus rec-assay, which we previously reported (9).
0
0,l
0.2
AF-2 Concentration (ps m 1 - l )
Figure 4. Effect of AF2 concentration to the sensor responses: (0) response of GY 5026 electrode; (0)response of GY 5027 electrode. The condition of measurements were same as shown in Figure 2 except for AF2 concentration.
cell as affected by AF-2 addition. However, cell number of GY5026 was not changed by the addition of AF-2. The difference in the effect of AF-2 addition between the two strains was due to the lysogenicity of GY5027. The addition of AF-2 caused the induction of phage lambda in GY5027, resulting in the lysis of GY5027. In other words, AF-2 caused the damage in GY5027 DNA and activated the Rec A protein. Therefore, by comparing the output current of the two electrodes, the mutagenicity of AF-2 was estimated. Effect of AF-2 Concentration to the Sensor Response. The effect of AF-2 concentration on the sensor response was then examined. Figure 4 shows the relationship between AF-2 concentration and the rate of current increase observed 2.5 h after addition of AF-2. With high concentration of AF-2, GY5026 has also shown some deficiencies in its respiration. This change was due to the nonspecific inactivation of respiration caused by the toxicity of AF-2 at high concentrations. The response of GY5027 shows a good correlation between AF-2 concentration and the rate of current increase, in the range of 0.01-0.2 pg mL-'. This linear correlation represents the dependency of AF-2 mutagenicity on AF-2 concentration. Application of the Sensor System to Other Mutagens and Chemicals. The estimation of mutagenicity of several mutagens by this sensor system was performed (see Table I). 4NQ0, mitomycin C, and MNNG were tested by this sensor system. The output current from the electrode immobilized GY5027 (lysogenicstrain) gradually increased while the output current from the electrode immobilized nonlysogenic strain GY5026 was not affected. In other words, each substrate showed the typical response to the mutagen. The sensitivity to each mutagen was superior to that of the sensor utilizing
It is also obvious in Table I that with the increase in mutagen concentration, the rate of the current increase of the lysogenic strain immobilized onto the electrode increases. In addition, the high concentration of such mutagens also produced the respiration deficiency in the nonlysogenic strain, as seen in the AF-2 measurement (see Figure 4). On the basis of these results, the mutagenicity of 4NQ0 and mitomycin C might be higher than that of MNNG. However, the comparison of substrate mutagenicity is very difficult because mutation depends on the rate and kind of reaction by each substrate with DNA. The responses of the sensor to nonmutagenic chemciais were then investigated, with the results summarized in Table I. Neomycin and kanamaycin are known antibiotics that inhibit protein synthesis. Benzalkonium chloride is a bactericide. When these chemicals were added to the test solution, the output currents from both electrodes increased. No significant difference in the response between GY5026 and GY5027 was observed. The current increase was due to the decrease in the number of viable cells immobilized onto the electrode. These results suggested that by using this sensor system, it is easy to detect the difference between mutagens and other toxic compounds. Moreau et al. reported that phage induction by a mutagen like aflatoxin B was observed within 60 min of incubation (IO). Ikeda et al. also reported that the addition of mitomycin C in a lysogenic strain caused phage induction within 90 min (11). The sensor system reported here, however, took more than 2 h to detect the induction. This difference in time required for the induction may be due to the minimum detectable cell number, as determined by the oxygen electrode. The previous methods are quite complicated because they require the separation of cell derivatives from the enzyme activity measurement solution or the preparation for microorganism incubation. Thus, this method requires a simple procedure, such that within 3-4 h of incubation reliable results can be obtained. In addition, we have recently developed a micro oxygen electrode using semiconductor fabrication technology (12). Application of this micro oxygen electrode to the mutagen detection system will yield a more simple and disposable type sensor system because of lower price and availability for mass production.
Anal. Chem. 1989, 61, 2391-2394
ACKNOWLEDGMENT The authors thank Ms. Eriberta N. Navera for help in compling this paper. LITERATURE CITED (1) Kada, T.; Turikawa, K.; Sadaie. Y., Mutat. Res. 1972, 16, 165-174. (2) A M s , B. N.; Lee, F. D.; Durston, W. E., R o c . Natl. Acad. Sci. U . S . A . 1973, 70, 782-786. (3) Ames, 6. N., Durston, W. E.; Yamasaki, E.; Lee, F. D.; R o c . Natl. A-d. Scl. U . S . A . 1973, 70, 2281-2285. (4) Bridges, B. A.; Mottershead, R. P.; Rothweii, M. A,; Green, M. H. L. Chem.-Bid. Interact. 1972, 5 , 77-84. (5) Uiltzur, S.; Weiser, 1.; Yannai, S. Mutat. Res. 1960, 74, 113-124. (6) Uiltzur, S.; Weiser, 1.; Yannai, S. Mutat. Res. 1961, 91, 443-450.
239 1
(7) Turner, A. P. F.; Karube, I.; Wiiison, G. S. Biosensors-Fundamentals and Applications; Oxford University Press: Oxford, 1987. (8) Karube, I.; Nakahara, T.; Matsunaga, T.; Suzuki, S. Anal. Chem. 1962, 54, 1725-1727. (9) Karube, I.; Nakahara, T.; Matsunaga, T.; Suzuki, S. Anal. Chem. 1961, 5 3 , 1024-1026. (10) Moreau, P.; Bailone, A.; Devoret, R. R o c . Natl. Acad. Sci. U . S . A . 1978, 73, 3700-3704. (11) Ikeda, H. Hen-igen to Dokusei 1979, 8 , 48-54. (12) Suzuki, S.; Tamiya, E.; Karube, I . Anal. Chem. 1968, 6 0 , 1078-1080.
RECEIVED for review November 14, 1988. Accepted August 17, 1989.
Characterization of Conducting Polymeric Stationary Phases and Electrochemically Controlled High-Performance Liquid Chromatography Hailin Ge and G. G. Wallace* Chemistry Department, Uniuersity of Wollongong, P.O. Box 1144, Wollongong, New South Wales 2500, Australia
A conductive polymer, polypyrroie containing dodecyi sulfate as counterlon, was electrosyntheslred on retkuiated vitreous carbon particles. The particles were packed into a column and the chromatographic performance of this new statlonary phase was evaluated. The use of potential control to Influence the Chromatographic behavior was investigated in a specially designed column. Mixed mode chromatographic behavlor was ldenwled during these experiments. Separation of the isomers m-toluic acid and p-toluic acid, as well as dimethyl phtaiate and dlethyi phthalate, indicates that useful seiecrtlvity can be obtained In this new stationary phase.
In previous work (1) carried out in these laboratories, the concept of using conducting polymers as chromatographic stationary phases was introduced. The use of conducting polymers enablea a convenient stationary phase synthesis route to be devised and also allows for electrochemical control of the stationary phase using an appropriately designed column. Conducting polymers may be synthesized either chemically or electrochemically according to
A number of polymers such as polypyrrole, polyaniline, polythiophene, polyfuran, as well as various derivatives can be produced in this way using either chemical or electrochemical oxidation ( 2 ) . Further flexibility in the design and synthesis of the stationary phase is available in that a range of counterions can be incorporated into these conducting polymers (2-8). Consequently, the mode of chromatography can be modified by employing appropriate monomers and counterions. An additional feature which makes the use of conducting polymers as chromatographic stationary phases attractive is the fact that the polymer properties can be altered electrochemically. For example, by switching the polymer from the conducting to less conducting state either chemically or 0003-2700/89/0361-2391$01.50/0
electrochemically,then counterions can be released from the polymer according to (2, 4-11)
This process also converts the polymer from a charged conductive state to a noncharged less conductive state. In the course of this work, new procedures for preparing conducting polymer chromatographic stationary phases have been developed. This procedure can be used to coat either conductive or nonconductive particles. A polypy-rroldodecyl sulfate polymer has been prepared and the chromatographic properties investigated and compared with commercially available columns. A new electrochemically controlled column has also been produced and employed to study the effects of the applied potential on this new stationary phase. Electrochromatographyhas been employed previously (12), however mostly using only carbon based particles. Some workers (13,14) have investigated the possibility of attaching modifiers to the carbon-based materials to modify chromatographic selectivity. The use of conducting polymers allows the properties of the stationary phase to be altered more markedly with electrochromatography.
EXPERIMENTAL SECTION Reagents and Materials. All reagents were analytical reagent (AR) grade unless otherwise stated. Pyrrole (Fluka, LR grade) was distilled before used. The solution employed in the electrochemical polymerization procedure was 0.2 M pyrrole and 0.1 M sodium dodecyl sulfate (SDS) in water. Dimethyl phthalate, diethyl phthalate, naphthalene, anthracene, benzoic acid, and m-toluic acid were 30 ppm while theophylline and caffeine were 10 ppm. All samples for injection were prepared in the mobile phase. Reticulated vitreous carbon (RVC) obtained from Energy Research Generation, Inc., was crushed to
