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Anal. Chem. 1989, 61, 1656-1660
Identification of Immobilized Bacteria by Aminopeptidase Profiling K. D. Hughes and F. E. Lytle* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
D. M.Huber* Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907
Conventional amlnopeptldase methods require cell concentrations of approxlmately 10’ to 10“ cells/mL and an Incubation period in the labeled substrates of 20 h. I n order to obtain the large number of cells requlred to perform this assay, a 36-48-h growth period must precede the assay. An improved procedure Is described that combines thne-resohred fluorometry and a nondestructive whole cell lmmoblllzatlon procedure. This method has reduced cell concentrations 80000-fold from that requlred for the standard assay, has reduced the room-temperature incubation period from 20 h to 3 mln, and has shortened the total turnaround time for the assay from 2.5 days to approximately 3-7 h.
At present a major goal of “improved” microbial identification techniques is to reduce the time required for identification. The turnaround time can be broken into two specific elements. The first is the growth period required to isolate and obtain enough sample to analyze, and the second is the time required to process the sample, including measurement and data reduction steps. The objectives of this research have thus been 2-fold: first, to reduce the total number of cells required to perform the assay and then to reduce the required incubation time of the microbial cells in the labeled substrates. Decreasing the number of cells and the incubation time period will decrease the total turnaround time for pathogen identification, and aminopeptidase profiling thus will become a much more practical method for bacterial and fungal identification. Identification methods that utilize inherent enzyme reactions in the organism can provide a “measurement advantage” due to the chemical amplification that is present with such systems. It is apparent then that bacterial identification schemes based on such measurements may have the most success in reducing the “total” analysis time. One such technique is aminopeptidase profiling, which has been employed since the early 1970’s to elucidate the metabolic Characteristics of bacterial pathogens (I). The method is based upon microbial metabolism of nonfluorescent L-amino acid P-naphthylamide substrates to produce the highly fluorescent tag, @-naphthylamine(PNA). Histograms that plot the normalized hydrolysis of these substrates provide a distinct aminopeptidase profile pattern that reveals various metabolic characteristics of that microbial species. This metabolic “fingerprint” can be utilized as a means for identification and differentiation of pathogen species ( 2 ) . Conventional aminopeptidase techniques utilize a filter fluorometer and as a result require cell concentrations of lo8 to 1O’O cells/mL and a 20-h incubation period to produce a significant @NAfluorescent signal. These high cell concentrations and lengthy incubation periods are due to the poor detection limit of the instrumentation and the large background emission associated with the assay. The lower limit of detection with the filter fluorometer is approximately io-’
M PNA, and the assay has a blank equivalent to 3 X lo4 M DNA (3) (Table I). The linear dynamic range of the assay is 1.5 orders of magnitude, and thus differentiation of closely related species is impossible even with the significant concentration of the bacterjal cells employed. In order to improve the assay to current standards, many modifications had to be implemented with respect to the instrumentation and the chemical procedures. The instrumental modification consisted of replacing the filter fluorometer with a time-resolved laser fluorometer. Typically, increases in source intensity without a simultaneous decrease in the blank have little effect on detection limits. Because of this, a detailed examination into reducing the magnitude of the interference from each component of the blank was investigated. The results of this study have been published by Coburn et al. ( 2 , 3 ) . By application of laser-based instrumentation to the assay, the detection limit for DNA has been reduced to nanomolar concentrations and the linear dynamic range of the assay is increased to 3 orders of magnitude. This improvement in measured signal to noise ratio has allowed for a decrease in the incubation period from 20 to 4 h when the same concentration of sample is used. Even with this five-fold reduction in the incubation period, there is a significant contribution to the blank from the autohydrolysisof the labeled substrates ( 2 , 3 ) . In addition, as a result of each substrate decomposing at a different rate and each microbial sample having a different inherent luminescence, a series of blank measurements are also required to calculate the amount of enzymatic hydrolysis. The ability to reduce cell concentrations for the assay hinges upon the extent of enzymatic hydrolysis observed for a particular cell concentration and the actual identification procedure. The standard aminopeptidase procedure consists of incubating a series of test tubes containing a suspension of cells and one of the amino acid substrates. The total number of cells required to produced an aminopeptidase profile is dependent on the concentration of cells and the number of substrates included in the profile. For example, in order to produce a 20-nutrient profile with one repetition, 40 5-mL tubes, each containing 5 X lo’ cells, or a total of 2 X lo9 bacterial cells would be required. In an attempt to reduce the total number of cells required and eliminate many of the cumbersome procedural steps in the method, the possibility of immobilizing the sample was explored. There have been many applications in the literature in which whole cells have been immobilized to allow for repeated use. Immobilization is completed by either chemical or physical processes, which include confinement by hydrophobic gels and adsorptionto polymers (4,5). Gel confinement techniques have diffusional limitations and thus are not suitable for a rapid aminopeptidase profiling technique. Physical adsorption to ion-exchange resins was attempted, and absorption of Pseudomonas phuseolicola cells was successfully completed. The anion-exchange resin, however, also absorbed the amino acid substrates and the fluorophore tag PNA. As a result, these investigations were terminated. Alternatively,
0003-2700/89/0361-1656$01.50/0 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 61. NO. 15. AUGUST 1, 1989
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Table I. Blank Contributions in Equivalent Concentrations of BNA
blank contribn
immobilized convention- time-resolved and ale assay only time-resolved
buffer autohydrolysis’ cell emission membrane apparatus
1.1 X M 2.4 X 2.5 X 10“ 5 X lO* 3.0 X 10” 3 X 10”
total
3 x 10-8
3 . 5 x 10“
M 2.4 X 1W” M 5Xl0-8 5 x 10-’0 5 x 10*
‘Data taken from ref 3. bCanventionaland time-resolved substrate concentration is 1 X lo6 M. Immobilized substrate concentration is 1 x lo4 M.
Figure 1. Miniature immobilization apparatus.
the use of micropore filters to remove bacteria from incubation solutions has appeared frequently in the literature as a simple and clean method of terminating enzymatic reactions and/or collecting fermentation products (6,7).The primary practical problem in pursuing this possibility was finding a membrane system convenient to use and one that adds little fluorescence to the blank through solvent-extractable contaminants. The system that evolved is a cell collection device housing a replaceable 25-mm membrane. The method developed is nondestructive to the sample and has the ability to differentiate between viable and nonviable cultures. Sample preparation is extremely simple when compared to other techniques, as there is no complicated procedure for cell lysis, biomarker extraction, or derivatization. The current methodology also has the capability of easily being automated compared to the original profiling procedure. Along with these characteristics, the modified procedures have reduced cell concentrations for the standard assay over 800000-fold and 800-fold from previously reported work (2), has reduced the room-temperature incubation period from 20 h to 3 min, and has shortened the total turnaround time for the assay from 2.5 days to approximately 3-7 h. EXPERIMENTAL SECTION Instrumentation. A detailed desaiption of the time-resolved laser fluorometer has been published previously by Cobum et al. (2). The excitation source is a PAR Model 2100 nitrogen laser. The sample is excited from the bottom in order to reduce scatked radiation, and the fluorescentsignal is collected at 9 0 O and focused into a JY single monochromater. The detector is an RCA 931A photomultiplier tube, and temporal resolution is obtained with a PAR Model 162/165 boxcar integrator. The integrated signal is subsequently analog to digital (A/D) converted and stored in an IBM XT. Immobilization Apparatus. A “large” immobilization apparatus was constructed from a cell collection device obtained from Ace Scientific (East Brunswick, NJ) and a Teflon insert obtained from a Millipore syringe fdter holder. The sintered glass membrane filter support originally in the cell collection device was removed and replaced with the Teflon insert. This modificationincreased the flow rate of the fdtration step and decreased the carryover between substrate incubations. Immobilization is achieved with a replaceable Durapore (HVLP) membrane filter with a pore diameter of 0.45 Pm. This system required an incubation solution of approximately 1 mL and a wash solution, which wa$ used to rinse the sample and dilute the incubation solution to measurement volume, of 4 mL. A miniature immobilization apparatus was prepared by cementing a small disposable high-performance liquid chromatography (HPLC) nylon syringe filter (Micron Separations, Inc.) onto a cut Pasteur pipet (Figure 1). The cement used was Duco cement manufactured by Du Pont. The syringe filter had a 3-mm
diameter and 0 . 4 5 pore ~ size, and contained a membrane made of nylon. Each of these was used for one bacterial sample and then discarded. The incubation solution has been reduced to 100 fiL while the wash and dilution volume has been reduced to 700 PL. Physical Immobilization. Quaternary ammonium exchange resin was incubated in 1 M NaOH for 24 b and then rinsed to pH 7 with distilled water. Physical adsorption of Pseudomonas phaseolicoln cells was completed by incubating, at room temperature, 1 g of the prepared resin and a 30% transmission (540 nm) suspension of the bacterial cells. Approximately los cells were adsorbed at the end of 90 min. This value correspondswell with published literature (4). Chemicals. Water was purified by distillation and stored in glass. The buffer consisted of 0.005 M Tham standard grade obtained from Fisher and 0.005 M NaCl analytical reagent grade obtained from Mallinckrodt. The buffer was acidified to pH 8 with concentrated HCI and stored in glass. The anion-exchange resin, Amherlite IRA-400, was obtained from Mallinckrodt. The amino acid 8-naphthylamidesubstrates were obtained from Sigma and stored as solids below 0 “C. The 8-naphthylamidesused were L-alanyl (ALA),L-arginyl (ARG), Ircystinyl-di- (CYS),Irglycyl (GLY),&histidyl (HIS),L-bydroxyprolyl (HPRO),L-leucyl (LEU), and Irlysyl (LYS). Stock solutions were prepared with Burdick and Jackson methanol at a concentration of 0.001 M. These solutions were stored at 4 “C and were stable for months Working solutions were prepared just prior to use at a 1:lO dilution. Biological Samples. Unless otherwise stated, cells were removed from 15 bold nutrient agar cultures and diluted in buffer. Microliters of this cell suspension were subsequently applied to the membrane immediately before analysis. All cells were trapped, but the fraction of viable cells in the original sample was not determined. RESULTS AND DISCUSSION The assay blank consists of Raman and Rayleigb scatter, emission from the buffer, a contamination of free DNA produced form the autohydrolysis of the substrates, and an intrinsic luminescence due to the biological matrix. Table I outlines the improvements afforded by the new measurement protocol. A change in buffer from Tris to Tham (same molecule, different name and purity) resulted in the fluorescence impurities being reduced 2 orders of magnitude. Preparing substrate stock solutions in methanol instead of water reduced the autohydrolysis by an order of magnitude (3). The introduction of temporal resolution reduced the interferences from scatter and the biological matrix, and this, combined with the above-mentioned fluorescent impurity reductions, allowed for a net decrease in the conventional aminopeptidase assay blank of 2 orders of magnitude. The methodology utilizing these procedural modifications was used to differentiate between races of P. megasperma sojae (3). Further improvement in the linear dynamic range of the measurement is theoretically available by reducing the cell concentration. This was attempted for cultures of Pseudomonas phaseolicola and Xnnthomonas phaseoli at
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989 037 03 0351 03 t
025-
0251 s o 2 I 015;
ALA
ARG
CYS
GLY
HIS
HPRO LEU
LYS
SUBSTRATE Flgure 3. Aminopeptidase profile of Pseudomonas phaseoko4 obtained with a filter fluorometer. A concentration of lo* cells/mL and a 20-h incubation period were utilized.
capability of hydrolyzing different amino acids. The stimulation of certain aminopeptidase enzymes early on in the profile could in theory alter substrates appearing later in the profile. The identity of the substrates and the order in which they were used in this investigation were chosen at random. The precision of the technique is illustrated with error bars in Figure 2, and since pattern recognition algorithms are used to match profiles, a pooled standard deviation for the substrates is calculated. The pooled standard deviation for the replicate profiles shown in Figure 2 is 2.4%. The reproducibility of the technique was examined further by comparing profiles produced on the same day from the same cells and profiles produced on different days with different cells. The mean character difference (9))which is similar to a Euclidean distance, was 1% for the same-day profiles. The mean character difference for profiles produced on different days is approximately 3 % . Reproducibilityof filter fluorometer profiles is slightly less, aa the standard deviation per substrate is approximately 4%. An aminopeptidase profile for Pseudomonas phaseolicola, obtained with a filter fluorometer, is presented in Figure 3 for comparison. This profile is produced with cell concentrations of lo8 cells/mL and a 20-h incubation period. This profile compares well with that shown in Figure 2 when kinetic variations due to cell concentration and incubation time are taken into account. The aminopeptidase technique is selfleveling, and thus substrates showing weak activity initially, such as leucine and lysine, tend to overtake other substrates when the incubation period is increased. This effect can be observed when the magnitude of hydrolysis for these two substrates is compared after 3 min and 20 h. There is greater lysine and leucine activity after 20 h, but the ratio between the two substrates remains the same for both incubation periods. It should also be noted that two of the major limitations to obtaining reproducible profiles with the standard technique are the variations in cell concentrations between each substrate incubation and percentage of viable cells present during the incubation. By utilization of the same sample of cells for each substrate incubation, much of the variation observed in the substrate hydrolysis within a profile and with replicate profiles can be eliminated. Nondestructive analysis of a sample can also be of great benefit when one is investigating long-term sample behavior. It is well-known that the composition of the growth media used to culture the bacterial populations affects both the identity and quantity of different cellular components. The adaptive behavior of Pseudomonas phaseolicola cells retained on the immobilization membrane was investigated (Figure 4). Each profile was produced with the same 5 x lo6 cells and a 3-min incubation. The introduction of a different culture medium (Pseudomonas Agar-F (PAG)) from that utilized in Figure 2 produces an aminopeptidase profile that lacks hydroxyproline activity (see Figure 4A). The incubation of these same cells 1 h later reveals that the cells have adapted to the substrates
ANALYTICAL CHEMISTRY, VOL. 61,NO. 15, AUGUST 1, 1989
A.
ALA ARG CYS
GLY
A.
HIS HPRO LEU LYS
ALA ARG
CYS GLY
HIS HPRO LEU LYS
8.
ALA ARG
CYS
GLY HIS HPRO LEU SU BST R AT E
1650
B.
LYS
Flgure 4. Aminopeptidase profiles of Pseudomonas phaseolicole showing adaptive behavior to the substrates used In the analysis: (A) initial and (B) 1 h later.
that were introduced in the first profile run. The hydroxyproline activity has increased, as seen in Figure 4B. The question of which profile is correct (Figure 4A or 4B) is of critical importance since pattern recognition algorithms are utilized for identification. At present, all identification techniques that rely on “fingerprints” of microbial organisms must obey strict guidelines with respect to culture media composition. The correct profile will correspond directly to the identity of the “standard” growth media. This observation is under further investigation as it may produce a method of eliminating the restrictions on growth media just discussed. A number of different growth media are being investigated to examine the aminopeptidase activity that results after an initial incubation with all the substrates. The ramifications of this investigation could be of major importance to microbial identification methods. Immobilization Apparatus Miniaturization. The drive to increase sensitivity and produce an assay capable of automation requires not only a further reduction in cell numbers but a reduction in reagent and measurement volumes. The immobilization system used in the investigations that produced Figures 2 and 4 utilized a 2 5 replaceable membrane. This system required an incubation solution of approximately 1mL and a wash solution, which was used to rinse the sample and dilute the incubation solution to measurement volume, of 4 mL. In order to reduce the solution volumes, a miniature immobilization system was created. This immobilization system employs a 3-mm membrane with a 0.45-fim pore size (Figure 1). The incubation solution can now be reduced to 100 HLwhile the wash and dilution volume has been reduced to 700 HL. This system was used to differentiate clearly both qualitatively and quantitatively between samples of Pseudomonas phaseolicola and Pseudomonas aureofaciens, two related species (Figure 5). Each profile was produced with 5 X los cells and a 3-min incubation period. It should be noted that the profile obtained from the Pseudomonas phaseolicola sample deviates with respect to the weaker hydrolyzed substrates from the profiles shown in Figure 2. This deviation is due to the decrease in cell concentration from that used in Figure 2. This situation can be corrected by increasing the number of viable cells immobilized or by increasing the incubation time period. The need for the presence of these substrates showing weaker activity must be appraised with respect to the ability of pattern recognition algorithms to identify the organism and the turnaround time for the iden-
SUBSTRATE Flgure 5. Aminopeptidase profiles obtained from 5 X lo5 cells and a 3-min incubation: (A) Pseudomonas phaseolicoia and (B) Pseudomonas aureofaciens .
~
Table 11. Comparison of Methods for an Eight-Nutrient Profile
time-resolved
immobilized
parameter
conventional assay
only”
time-resolved
cell growth no. cells req tot incubn time no. of measmts total time
36-48 h 4 x 109 20 h 16 2.5 days
2-6h 50 x 106 4h 16 6-10 h
2-6 h 500 x 103 24 min 8 3-7 h
and
‘Data taken from ref 2.
tificaiton. If identification can be completed without the presence of the weaker activity substrates, then further reductions in cell concentrations can be achieved. These considerations are being investigated further. Present and Predicted Performance. The conventional technique and the technique described in this paper are compared in Table 11. The growth period that precedes the assay has been reduced from 36-48 to 2-6 as a direct result of the total number of cells required to perform the assay being reduced from 4 X lo9to 5 X lo5. The autohydrolysis values represent the contribution to the blank that occurs during the full incubation period cited. The incubation time of each substrate has been reduced from 20 h to 3 min due to the large increase in signal to noise ratio facilitated by utilizing an immobilized cell technique. For an eight-nutrient profile, the total incubation time is 24 min since each substrate is sequentially instead of the conventional simultaneousincubation of each substrate. It is obvious, though, that all eight substrates are not needed to differentiate between these two species; thus, the analysis time could be reduced accordingly. One other important outcome of the immobilization procedure is that the number of sample measurements required to perform the assay has been reduced by a factor of 2. This is a consequence of the assay having a constant blank reading for each substrate that does not change with bacteria type or concentration. The end result of these instrumental and methodological improvements can be seen by the decrease in total turnaround time for identification from 2.5 days to approximately 3-7 h. Research is under way to increase the sensitivity of the technique and lower the cell numbers still further. The av-
Anal. Chem. 1989, 61, 1660-1666
1660
enues being explored consist of decreasing reagent and dilution volumes and improving the instrumental sensitivity and selectivity by incorporating improved optics and a nanosecond transient recorder. Analysis is also under way that will unveil the experimental trades present between the cell concentration, the substrate concentration, and the incubation time period. Optimization of the procedure will undoubtedly allow for a decrease in cell concentrations with a minimal increase in incubation time. The end goal of these improvements is total elimination of the cell growth period. Registry No. Ala-P-naphthylamide, 720-82-1; Arg-Pnaphthylamide, 7182-70-9; di-Cys-8-naphthylamide, 1259-69-4; Gly-fl-naphthylamide,716-94-9;His-8-naphthylamide,7424-15-9; Hpro-fl-naphthylamide,3326-64-5;Leu-8-naphthylamide,732-85-4; Lys-P-naphthylamide, 4420-88-6;aminopeptidase, 9031-94-1.
LITERATURE CITED (1) Huber, D. M.; Mulanax, M. W. Phyfopathdogy 196S, 59, 1032. (2) Coburn, J. T.; Lytle, F. E.; Huber, D. M. Anal. & e m . 1985, 5 7 , 1669.
(3) Coburn, J. T.: Lytle, F. E.; Huber, D. M. Anal. Blochem. 1986, 154. 305. (4) Vieth, W. R.; Venkatsubramanian, V. I n Immobksd Mlwabiel cells; Venkatsubrarnanian, V., Ed.; ACS Symposium Series 108; American Chemical Society: Washington, DC, 1979; Chapter 1. (5) Rossomando, E. F. High Performance Liquid Chromatographyin Enzymatic Ana/ysls; John Wiby & Sons: New York, 1987. (6) Pau, C.; Patonay, G.; Moss, C. W.; Hoiiis, D.; Carlone, G. M.; Plikaytls, 0. D.; Warner, I . M. Clin. Chem. lS87. 33, 337. (7) Gabler. R.; Ryan, M. I n PuMcatEOn of Fementafion products; LeRolth, D., Ed.; ACS Symposium Serbs 271; American Chemical Society: Washington, DC, 1985; Chapter 1. (8) Coburn, J. T.; Forbes, R. A.; Freiser, 8. S.; Eecter, L.; Lytle, F. E.; Huber, D. M. Anal. Chlm. Acta 1986, 184, 65-76. (9) Jurs, P. C.; Isenhour, T. L. Chemica/App/icationsof Pattern Recognition; Wiley: New York, 1975.
RECEIVED for review February 15,1989. Accepted May 3,1989. This research has been supported by the National Science Foundation Grant CHE-8320158 and the Showalter Foundation. K.D.H. is also grateful to the Purdue Research Foundation for a David Ross Fellowship.
Water as a Unique Medium for Thermal Lens Measurements Mladen Frankol a n d Chieu D. Tran* Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53233
The temperature effect on thermal lens measurements In water has been Investlgated. The magnitude and slgn of the thermal lens signal lntenslty were found to be strongly dependent on the temperature of the aqueous solutlon. Dependlng on whether the measurements are performed at temperatures lower or hlgher than -0.01 O C , the photoinduced thermal lens can have either a poSnlve (converglng) or negative (dhrerglng) focal length. At precbly -0.01 f 0.04 OC, no thermal lens dgnal could be observed. TMS is because the dn/dTvalues of water are poeltlve at T < -0.01 OC, negatke at T > -0.01 O C , and equal to zero (Le., maxlmum refractive index) at T = -0.01 O C . Thls unique characterlstlc was exploited to enhance the senSnlvlty of thermal lens measurements In water. For Instance, the thermal lens a n a l of an aqueous solution was enhanced up to 2.4 times when the temperature of the solution was Increased from +20.0 to HO.0 OC. For thermally unstable compounds, the sensltklty enhancement was achleved by synergldc use of the bimodal characterktlc of the thermal lens technique and the temperature effect on the thermooptlcal properlies of water. Typlcab, two sample cells, one at -7.9 O C and the other at +12.O O C , were placed on both sldes and symmetrically about the beam walst. The sensltlvlty of this two-cell system was 1.80 or (1 T ) tlmes that of the slngk cell ( T Is the transmittance of the flrst cell).
+
Thermal lens techniques have been demonstrated to be a sensitive method for low-absorbance measurements (1-1.2). Absorptivities as low as lo-' have been measured by using these techniques. The technique is based on the nonuniform temperature rise that is produced in an illuminated sample
* To whom correspondence should be addressed. Permanent address: Nuclear Chemistry Section, J. Stefan Institute, Ljubljana, Yugoslavia.
by nonradiative relaxation of the energy absorbed from a TEMm laser beam (1-12). For weak absorbing species, the thermal lens signal, which is measured as the relative change in the laser beam center intensity in the far field, AIh/Ih, is related to the excitation laser power P and sample absorbance A by
AIbc 1.21P(dn/dT)A - Ibe
Xk
where X is the wavelength, and dn/dT and k are the temperature coefficient of the refractive index and thermal conductivity of the solvent, respectively (I,2 ) . I t is thus clear that in addition to the sample absorbance and excitation laser power, the thermal lens signal intensity is directly proportional to dn/dT and inversely proportional to the k value of the solvent. Generally, nonpolar solvents provide good media for thermal lens measurements owing to their high dn/dT and low k values (1,2,10,12).Conversely,water, which is the most powerful and widely used solvent in spectrochemical analysis, specifically for metal ions and biological compounds, is considered to be the worst medium for thermooptical techniques because it has very low dn/dT and high k values (I, 2,10,12). This is very unfortunate because it severely limits the scope of these techniques. As a consequence, considerable efforts have been made in the last few years to ameliorate the thermal physical properties of water. Most notable methods include the use of micelles or reversed micelles to enhance the sensitivity of thermal lens measurements in water (10, 12). Unfortunately, in spite of the success, these methods involve the addition of surfactants into the aqueous solution, which sometimes may produce some unwanted effects (13-15). It is thus particularly important that a new method which can improve the thermal physical properties of water without the use of any additive be developed. The extensive hydrogen-bonding network and multiple structural characteristics enable water to be unique among solvents. For instance, its density increases as temperature
0003-2700/89/0361-1660$01.50/0 0 1989 American Chemical Society
