1925
Anal. Chem. 1985, 57, 1925-1928
4'20 4.70
i
,a
SURF ACE
PH 6 20 6 70
7 20 IO
01
100
1000
PENICILLIN (mM) Flgure 1. Experimental (full points) and theoretical (open points) calibration curve for a 100 pm penicillin ENFET: (a) 0.02 M pH 7.2 phosphate buffer in 0.14 M NaCI; (b) 0.08 M buffer. I 00.00%
/'/
80.00%/
x
FULL
60.00%
RESPONSE 40.00%
*-•
1 /c
I1 0
20
40 60
80
100 I20 140 160
TIME ( W C ) Flgure 2. Experimental (open points) and theoretical (full points) time response of 100 pm penicillln ENFET concentration step, 0 to 10 mM benzylpenicillin, 0.02 M phosphate buffer pH 7.2 in 0.14 M NaCI.
EXPERIMENTAL SECTION The penicillin EIWETs and reference transistors were prepared by the same procedure as described in part 2 for glucose EIWET. Penicillinase (Sigma, P0389) was derivatized with methacrylate group and covalently bound to the polyacrylamide matrix. All measurementswere done in buffered 0.14 M NaCl at 33 O C . The buffer concentrations were chosen such that they corresponded to the conditions used in our first penicillin paper (5). The concentration measurements were done by diluting a 100 mM stock solution of sodium benzylpenicillin (Sigma, PEN-NA).
RESULTS AND DISCUSSION The experimental and calculated response of a typical 100 pm penicillin ENFET is shown in Figure 1. It is interesting to note that the maximum slope of these curves is lower (two pH/decade of substrate concentration) than that for the one-substrate, one-buffer case (part 1). This is due to the additional titratable group on the penicilloic acid (pK, = 5.2) which effectively captures the hydrogen ions at pH below 6.2. This means that the detection limit for the penicillin case is the same as that for the one-buffer case. Clearly, the dynamic range of this sensor is also narrower because of the capture of the enzymaticallygenerated hydrogen ions at low pH values (high substrate concentration). The time response (Figure 2) for the penicillin ENFET is similar to the one-buffer case (part 1) and not too different from that obtained with out previously reported penicillin ENFET (5). CONCLUSIONS The model which has been proposed for the penicillin ENFET agrees with the experimental data. It confirms previous experimental observations (2-4)and theoretical analysis (part 1)which shows that the buffer capacity is the single most important parameter governing the response of pH-based enzymatic sensors. It is shown that the buffer can be either an independent component of the system and/or a buffer moiety related to one or more species in the enzymatic reaction itself. In the case of benzylpenicilloic acid it is its second weakly acidic group. It is again shown that the unit slope has no special meaning with these sensors. The detection limit of the penicillin ENFET probe is well above the penicillin levels found in biological fluids during the penicillin therapy. It is possible that this sensor may fiid its application in monitoring of the fermentation production of penicillins. Registry No. Penicillin, 1406-05-9;benzylpenicillin, 61-33-6. LITERATURE CITED (1) Waley, S. G. Blochem. J . 1975, 149, 547-551. (2) Papariello, G.;Mukherji, A.; Shearer, C. M. Anal. Chem. 1973, 45,
790-792. (3) Nilsson, H.; Mosbach, K. Blofechnol. Bloeng. 1078, 20, 527-539. (4) Niisson, H.; Akerlund, A.; Mosbach. K. Blochlm. Blophys. Acta 1973, 320, 529-534. ( 5 ) Cares, S.: Janata, J. Anal. Chem. 1080, 52, 1935-1937.
RECEIVED for review January 22,1985. Accepted April 5,1985. This work has been supported by a grant from NIGMS, Grant NO. GM 22952-08.
Determination of Total Protein in Serum Using a Tyrosinase Enzyme Electrode Takashi Toyota, Shia S. Kuan, and George G. Guilbault* Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148 A novel enzymatlc, eiectrochemlcal method has been developed for the simple, reilable, and accurate analysls of total protein in serum. The protein In serum Is flrst hydrolyzed wlth pepsin and the tyroslne cleaved Is subsequently determined by a tyroslne selective electrode. When compared with the biuret method used In the hospital, a correlation coefflclent of 0.970 was obtained. Thus, the method appears to be feaslbie and practical for the analysis of total serum protein In clinical laboratories. 0003-2700/85/0357-1925$01.50/0
The total serum protein of healthy young and middle aged adults is found to be 6.0 to 8.0 g/dL when recumbent, but 6.5 to 8.5 g/dL when ambulatory (1). During dehydration, or in cases of multiple myeloma, the total protein may reach to 10 g/dL or higher. On the other hand, in hypoproteinemia, the total serum protein level drops below 6.0 g/dL. Several methods have been developed for the determination of total protein in serum, including the classical Kjeldahl method (2), the biuret method (3), the Folin-Ciocalteau 0 1985 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
reagent procedure (4), and the refractive index method (5). The Kjeldahl method is widely used and accepted as a standard method but is too tedious and time-consuming to be used as a routine assay procedure. The commonly used biuret method, suffers from low sensitivity and turbidity problems. The Folin-Ciocalteu procedure is 5 to 10 times more sensitive than the biuret reaction, but it is subject to several interferences originating from the sample matrix. Rechnitz (6) described the first electrochemical method for protein assay using a silver sulfide membrane electrode to measure the free sulfhydryl group liberated by alkaline denaturation of the protein. This method also suffers from many interference problems. An alternative approach appears to be the use of an enzyme which selectively hydrolyzes peptide bonds of the protein. The amino acid moieties cleaved are subsequently quantitated by an amino acid selective electrode. A tyrosine selective enzyme electrode has been developed by several workers wing immobilized L-tyrosine decarboxylase (7-10). Rechnitz, with the use of an NH3 liberating Aeromonas phenologenes species (ATCC29063), developed an induced bacterial electrode for the measurement of tyrosine using NH3 gas electrodes as sensors (11). Drawbacks of both the decarboxylase and the bacterial electrodes are slow response and recovery times. Because the activity of commercially available decarboxylases is very low (0.3-1.0 U per mg of solid), a large amount of enzyme (over 8 mg) must be immobilized on the membrane for better reactivity, resulting in a thick immobilized enzyme layer. In order to overcome problems of poor specificity, low sensitivity, and slow response characteristics, we used a tyrosinase electrode to monitor amperometrically the concentration of tyrosine moiety liberated from the hydrolysis of serum protein by pepsin(EC 3.4.23.1). The amperometricbased electrode has better sensitivity as well as faster response and recovery times than the previously described gas-sensing probes. The proposed method provides a simple, convenient, and reliable measurement of total protein in serum. EXPERIMENTAL SECTION Apparatus. All amperometric measurements were performed with a Radiometer PHM72MK2 digital acid-base analyzer and a Radiometer REC 61 servograph recorder. The O2electrode used was a Radiometer E5047, fitted with a polypropylene membrane, a s m d intestine membrane obtained from Universal Sensors (New Orleans, LA), thickness 15pm and 20 m,and a dialysis membrane (a thin collagen membrane, 20 pm from Center Technique du Cuir, Lyon, France). Solutions in the reaction cell were mixed using a ministirrer at 600 rpm controlled by an EC Molomatic motor control stirrer (Model E550M, Electrochart Co., Hopkins, MN). A water-bath shaker (Eberbath Co., Ann Arbor, MI) was used for the enzymatic hydrolysis of samples in small test tubes. A Fluoromicrophotometer (AmericanInstrument Co., Silver Springs, MD) was employed for the assay of tyrosine liberated during preliminary studies. Reagents and Materials. The tyrosinase (2600 U/mg, from mushroom), pepsin (2900 U/mg, from porcine stomach mucosa), a-chymotrypsin (EC 3.4.21.1, Type 11, 48 U/mg, from bovine pancreas), amino acids, bovine albumin (BA), glutaraldehyde (25% solution), trichloroacetic acid, and 1-nitroso-2-naphtholwere obtained from S i a Chemical Co. (St.Louis). A standard protein working solution was prepared by dissolving one part of human albumin and one part of human globulin in 0.5% NaCl solution to a final concentration of 10 g of protein/dL. Other chemicals used were analytical grade reagents. Procedure. a. Preparation of Tyrosinase Electrode. The basic procedure described for preparation of a tyrosine decarboxylase electrode was essentially the same as that described by Havas et al. with some modifications (IO). Place a polypropylene membrane and a wet small intestine membrane on top of the outer jacket of an O2electrode. Then, press an 0-rkgdown into a groove around the jacket. Treat the small intestine membrane first with 100 pL of a-chymotrypsinsolution (1mg/mL).
After washing and drying place 20 pL of 15% BSA solution on a small intestine membrane and mix with 2-3 mg of tyrosinase, blending with a nylon string. Then, add 1 pL of 25% glutaraldehyde and quickly mix the solution for 20-30 8. Store the jacket overnight in a refrigerator at 4 'C. The next day, cover the enzyme layer with a dialysis membrane and wash with Tris buffer (0.05 M, pH 7.0). Store the enzyme layer at 4 "C in Tris buffer when not in use. Nine tyrosine electrodes were carefully made and used in this study. The variations in reactivity of the freshly prepared electrodes were found to be within 10%. This electrode is now commercially available from Universal Sensors, b. Electrode Measurement. Immerse the electrode in 1.0 mL, 0.05 M, pH 8.0 Tris buffer saturated with O2by blowing air bubbles into the buffer prior to use. Then, add various volumes of standard tyrosine solution or serum hydrolysates to the buffer solution. Record the resulting current-time curves on a strip chart recorder and measure the initial rate of current change. Calculate the concentration of tyrosine from a calibration curve of the rate of current change vs. tyrosine concentration. c. Hydrolysis of Total Protein. Place 100 pL of standard protein solution or serum, 100 pL of pepsin (10 mg/mL) and 800 pL of 0.1 M glycine buffer (pH 2.0) in a glass tube covered with a paraffin film. Keep the tube in a water bath shaker at 37 'C and shake at moderate speed for 10 min. Mix the resulting hydrolysate in the tube immediately with 4 mL of Tris buffer (0.05 M, pH 8.0) and assay the concentration of tyrosine liberated using the tyrosinase electrode. Calculate the protein content of the sample by using a calibration curve. RESULTS AND DISCUSSION 1. Fluorometric Measurement in Preliminary Studies. We used the same procedure developed by Anbrose et al. (12) to assay tyrosine in this study. The fluorescence wm measured a t A, of 436 nm and a A, of 520 nm. A calibration curve of the concentration of L-tyrosine by the fluorometric measurement showed a linear dynamic range between 0.2 pg/mL M). The method (1.1 X lo4 M) and 2.0 pg/mL (1.1X is more sensitive than the electrochemical measurement, but the procedure is too Iaborous (over 40 min per assay) and the sample must be centrifuged prior to determination. 2. Enzymatic Hydrolysis Using Pepsin. Kalmar (13) investigated both multienzyme and single enzyme systems for the hydrolysis of casein and found pepsin catalyzed the rapid hydrolysis of casein (10 min) at low pH. The hydrolysis of BSA (5 mg/mL) with pepsin (1 mg/mL) in 0.1 M glycine (pH 2.0) indicated a maximum concentration of tyrosine is obtained in less than 5 min. If cy-chymotrypsin is used,hydrolysis to the same level requires 20 min. The effect of pH on the hydrolysis of BSA with pepsin was studied; the catalytic activity of pepsin decreases with increasing pH, having a maximum a t pH 2.0. 3. Performance of Tyrosine Electrode. Tyrosinase is known to catalyze the oxidation of tyrosine (14) to dopaquinone as shown in the following equation: tyrosine
+ O2
tyrosinase
dopaquinone
+ H202
(1)
An amperometric tyrosine electrode we developed is based on the measurment of O2uptake during the oxidation. When the electrode was set at the fixed potential, the current change observed is proportional to the tyrosine concentration present in the buffer solution. a. Response of Tyrosine Electrode. In a study of the response curves of the tyrosine electrode vs. various concentrations of L-tyrosine, it was found that the electrode reaches a steady state within 2 to 3 min after the addition of sample. However, the rate of initial current changes with increasing concentration of tyrosine, so this rate method was thus used to construct a calibration curve. The electrode returns to its original base line within 2-3 min after it was immersed in a fresh buffer solution with vigorous stirring. The relative standard deviation of the response was 1.8% for seven SUC-
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
1927
Table I. Relative Activity of the Tyrosinase Electrode toward Various Compounds compounds L-tyrosine L-cysteine L-phenylalanine L-serine L-alanine L-lysine L-glutamic acid L-arginine L-proline L-hydroxyproline L-leucine L-histidine L-methionine L-aspartic acid L-isoleucine L-tryptophan L-asparagine L-valine L-threonine L-glutamic acid
re1 activity, % 100 0 0 0 trace
trace trace trace 0 0 0 0 0 0 0 0 0 0 0 0
tyramine HC1 p-hydroxyphenylacetic acid
130 47
D-tyrosine
100
cessive measurements of a standard tyrosine solution (8.16 x 104 M). A calibration curve constructed for this tyrosinase electrode demonstrated the lowest limit of detection was 1.33 x lod M and the linearity extended up to 2.5 X lo4 M; above this concentration, deviation from linearity resulted. b. Effect of pH. A comparison of the relative activity of immobilized and soluble tyrosinase for the determination of tyrosine a t various pHs in 0.05 M Tris buffer was conducted. Maximum activity is obtained at pH 7.5 for both immobilized and soluble enzymes. Results showed that immobilized tyrosinase did not improve its pH profile because the relative activity at various pHs was almost the same as that with soluble enzyme. c. Specificity and Interference. As shown in Table I, the L-tyrosine electrode is very specific. Nineteen amino acids did not show any response, but structurally similar tyramine and p-hydroxyphenylacetic acid interfere with the response of this electrode. DTyrosine also reads because the tyrosinase used is a DL enzyme. However, the concentrations of these compounds in serum are insignificant (15)and hence do not affect the accuracy of total protein assay. d. Stability of Tyrosinase Electrode. Tyrosinase electrodes were prepared with various concentrations of GA (glutaraldehyde) and BSA (bovine albumin) in order to form very reactive and more stable enzyme layers over a long period of time. Generally, when the volume of GA was increased, the enzyme layer became more rigid, but did not help in stabilizing the activity. The concentration of BA affected the activity, and thus the volume used should be as small as possible. The optimum composition was found to be the following: 3 mg of tyrosinase, 1 pL of 25% GA, and 20 pL of 15% BA. The useful lifetime of an enzyme electrode depends mostly on the number of assays run, normally ranging from 15 to 20 days, with 10 to 20 assays/day (Figure 1). 4. Determination of Total Protein Using a Tyrosinase Electrode. Since serum proteins are composed of 60% albumin and 40% globulin, standard solutions (10 g/dL) containing 2 parts of human albumin and 1 part of human globulin were diluted with double distilled water to prepare 4,5,6,7,8, and 9 g/dL working standard solutions. An assay of total protein in these standard solutions was performed by an enzymatic hydrolysis with pepsin, and subsequent mea-
$
BO-
k! 5
IO
15
20
25
PERIOD ( DAY) Flgure 1. Stability of tyrosinase electrode.
Table 11. Total Protein of Standard Solutions Assayed by the Tyrosinase Electrode Method total protein, g/dL
rate of current,” pA/min
10.0 9.0 8.0 7.0 6.0 5.0 4.0
0.71 0.63 0.56 0.47 0.42 0.35 0.28
av recovery, %
*
99.0 2.9 99.0 f 2.0 97.0 i 7.0 101.0 2.2 100.0 f 2.7 96.0 f 7.1
*
98.7 f 4.0b
OAverage of three to five runs. Mean value. Table 111. Comparison Study of Total Protein in Serumb sample no. 1 2 3 4 5 6 7 8 9 10 11 12
total protein, d 1 0 0 mL hospital electrode
(n
6.1 6.3 7.1 5.5 7.0 4.8 7.3 7.7 5.0 7.5 8.1 5.7
(x).
6.0 6.0 6.8 5.8 7.2 4.8 7.2 7.5 5.3 7.6 8.2 5.7
a Ayerage of three to five runs. Linear regression analysis: P = 1.08X - 0.47, r = 0.976, u = 0.09. -
surement of tyrosine was conducted using the tyrosinase electrode. The average recoveries were between 96% and 101%)and the average standard deviation was 4% (Table 11). 5. Comparison Study. Twenty serum samples obtained from Touro Infirmary, New Orleans, LA, were analyzed for the total protein by the above method and compared with the biuret method used in the hospital laboratory. The correlation coefficient between the two methods was 0.976 and the linear regression line was y(hospita1) = l.O8~(electrode)- 0.47. Some of the data are shown in Table 111. The average standard deviation is excellent for 20 samples, each assayed an average of four times. Thus, the method developed appeared to be an accurate and reliable method for the assay of total protein in human serum. Registry No. Tyrosinase, 9002-10-2; L-tyrosine, 60-18-4.
LITERATURE CITED (1) Thompson, R. H.; Wootton, I. D. P. “Biochemical Disorders in Human Disease”, 3rd 4.;Academic Press: New York, 1970.
1928
Anal. Chem. 1985, 57, 1928-1930
(2) ArchlbaM, R. M. I n “Standard Methods of Clinical Chemistry”; Seieg son, D.,Ed.; Academic Press: New York, 1958;Vol. 2,p 91. (3) Doumas, 8. T.; Bayse, D. D.; Carter, R. J.; Peters, T., Jr.; Schaffer, R. Clln. Chem. (Winston-Salem, N.C.) 1981, 27, 1642-1645. (4) Variey, H. “Practical Clinical Biochemistry”, 4th ed.; Heinemann Medicai Books, Ltd.: London, and Interscience: New York, 1967. (5) Rubln, M. E.; Wolf, A. V. J. Biol. Chem. 1957, 225, 869-873. (6) Alexander, M.; Rechnitz, G. A. Anal. Chem. 1974, 46, 250-254,
860-865. (7) Gullbault, G. G.; Shu, F. R. Anal. Chem. 1972, 44, 2161-2165. (8) Berjonneau, A.-M.; Broun, T. D. Pafhol. Blol. 1974, 22, 497-502. (9) Calvot, C.; Berjonneau, A.-M.; Gellf, G.; Thomas, D. FEBS Left. 1975, 59,258-263. (IO) Havas, J.; Gullbault, G. G. Anal. Chem. 1982, 5 4 , 1991-1995.
(11) DiPaoiantomo, C. L.; Rachnitz. G. A. Anal. Chim. Acta 1982, 141, 1-10. (12) Anbrose, J. A.; Becker, R.; Blake, E.; Sldeman, L; Wainer, S. Clln. Chem. (Winston-Salem, N.C.) 1974, 20. 505-510. (13) Kalmar, A. D. M.S. Thesis, University of New Orleans, 1980. (14) Duekworth, H. W.; Coteman, J. E. J . Biol. Chem. 1970, ,245, 1613-1617. (15) Knox, W. E. “The Metabolic Basis of Inherited Disease”, 3rd ed.; Stanbury, J. E., Wyngaarden, J. E., Fredrickson, D. S., Eds.; McGrawHIII: New York, 1972;p 266.
RECEIVED for review January 30, 1985. Accepted April 11, 1985.
Solvent Perturbation Fluorescence Immunoassay Technique Clarke J. Halfman,* Franklin C. L. Wong, and Dennis W. Jay Department of Pathology, University of Health ScienceslThe Chicago Medical School, North Chicago, Illinois 60064
The use of fluorescent dyes to label analyte In llgand blndlng assays affords the posslbllty of convenlent, homogeneous assays. The homogeneous response depends upon a slgnlflcant difference In a fluorescent property of bound compared to free labeled analyte. We have found that dodecyl sulfate quenches the emlsslon lntenslty of free fluoresceln labeled gentamycln wlthout lnfluenclng the emlsslon lntenslty of labeled gentamlcln bound to gentamlcln antlbody. Thls preferentlal quenchlng by detergent Is demonstrated to serve as the basis for a homogeneous fluorescence Immunoassay lor gentamlcln requiring only slmple lntenstty measurements. The method may be used to measure other analytes when It can be demonstrated that the perturblng agent (In this case, detergent) preierentlally Influences the lntenslty of free labeled analyte. Thls preferentlal perturbation may be assured by judicious choke of perturblng agent and labellng fluor so that the lnteractlon between labeled analyte and the perturblng agent occurs with the analyte molety and not wlth the fluor molety.
Fluorescent dyes have proved practically useful as labels in ligand binding assays and provide a homogeneous response when a fluorescent property of bound labeled analyte is sufficiently different from that of free. Fluorescence intensity is the simplest property to measure, but significant intensity differences between bound and free labeled analyte do not often occur. Only for the case of fluorescein labeled thyroxine (I) has the bound/free intensity ratio ( 2.7) been reported to be sufficiently great so that measurement of intensity provided a useful, homogeneous response variable. Although a boundlfree intensity ratio of -0.8 has been reported (2) for fluorescein-labeled gentamicin, the degree of intensity difference does not seem sufficiently great to be practicably useful. A significantly greater fluorescence polarization for bound labeled analyte compared to that of free should, in principle, be a general phenomenon for analytes of sufficiently small molecular size (3), and fluorescence polarization has been demonstrated to provide a practicable, homogeneous response variable (4). Polarization, however, is a calculated parameter determined from measurements of both the vertical and horizontal emission components with polarizers in the exciN
0003-2700/85/0357-1~28$01.50/0
tation and emission beams. One of the polarizers must be rotated before each measurement, and the presence of each polarizer in the light beam reduces light intensity and signal strength by a factor of rI. Potential sensitivity is thus reduced by up to a factor of about 10. Because of the greater convenience and potential sensitivity of intensity measurements, efforts have been expended to cause a preferential alteration of intensity from bound or free labeled analyte. The principle of excitation energy transfer was exploited (5) by additionally labeling analyte-antibody with a nonfluorescent dye, the absorption spectrum of which overlaps the emission spectrum of the dye used to label analyte. The excitation energy of the analyte label (donor dye) is transferred to the antibody label (acceptor dye) when the two dyes are sufficiently proximate, resulting in decreased emission from donor dye. Energy transfer, and consequent quenching of donor dye emission, occurs only when labeled analyte is bound to antibody (labeled with acceptor dye). Emission from bound labeled analyte is thus preferentially quenched, and the assay response is increased intensity with increasing concentration of analyte from standards or specimen. One difficulty which this method presents results from the necessity to conjugate antibody with a relatively high mole ratio (-10-20) of acceptor dye to assure that a t least one acceptor dye molecule becomes conjugated sufficiently close to each hapten binding site. On the other hand, too high a degree of conjugation may diminish antibody affinity, so that an optimum degree of conjugation must be established for each antibody preparation. Another difficulty with this method is that the acceptor dye must have minimal fluorescence since it is present a t far higher concentrations in the assay tube than is the donor dye because of the high degree of conjugation and also because when antibody is labeled with acceptor dye, so are all the other serum proteins present in the antiserum unless the specific immunoglobulin is isolated. Another means to preferentially quench emission from free-labeled analyte employs an additional antibody to the dye label (6)and is based upon the observation that fluorescein emission is quenched when the dye is bound to its antibody (8). Steric hindrance prevents dye binding by the dye antibody when labeled analyte is bound to analyte antibody. Assay response is thus a decrease in intensity, with increasing concentrations of analyte from standards or specimen. A disadvantage of this method is the requirement for a second antibody to the label which must consistently be of high 0 1985 American Chemlcal Society
