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A study of interferences in glucose measurements in blood by hydrogen peroxide based glucose probes
ANALYTICAL
BKXIHEMISTRY
13, I 14-12 1 ( 1986)
A Study of Interferences in Glucose Measurements in Blood by Hydrogen Peroxide Based Glucose Probes GIUSEPPEPALLESCHI,~.' MOHAMMADALINABIRAHNI,-~GLENN J. NGEH NGWAINBI,~ANDGEORGEG.GUILBAULT*~~
J. LUBRANO,*
*Universal Sensors, Inc., 5258 Veterans Memorial Boulevard, Metairie, Louisiana 70006; and tDepartment of Chemistry, University of New Orleans, New Orleans, Louisiana 70148 Received April 24. I986 The main blood constituents which could interfere in clinical glucose measurements using a hydrogen peroxide based glucose electrode have been investigated using severaldifferent membranes and constant and sweeping potentials. Roth diluted and whole undiluted sera were investigated. With a 3500 molecular weight cut-off (MWCO) membrane, acetaminophen, cysteine, and ascorbic acid can interfere. With a 100 MWCO membrane, only acetaminophen interfered. Q 1986academic Press, Inc. KEY
WORDS: glucose electrode; interferences; vohammetry; membranes.
The importance of glucose determinations for the diagnosis and treatment of diabetes has been well recognized. Before the twentieth century, over 30 methods for the measurement of glucose in biological fluids were already proposed. The desire for simplicity, precision, and good specificity has brought about the development of several hundred methods and modifications within the past several decades alone. The current methods used to maintain a normal glucose level in treating diabetes require daily, weekly, or monthly analysis of serum or urine. The most reliable electrochemical system for the assay of glucose utilizes an enzyme system, glucose oxidase (EC 1.1.3.4) according to the reaction: /3-D(+)glucose + 02 + Hz0 ‘“-zdase Hz02 + gluconic acid An assay of the glucose level can be made electrochemically either by monitoring the oxygen consumption with a platinum cathode ’ Present address: Department of Science and Chemical Technologies, IInd University of Rome, Tor Vergata, Italy 00173. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproductton in any form reserved.
(1,2), or by monitoring the Hz02 produced with a platinum anode (3). With the O2 system one generally looks at a small rate of current change in the presence of a large current background. With the H202 system, one looks at a similar rate or current change but in the presence of a much smaller background. The purpose of this work was to investigate some of the blood constituents which could interfere in clinical glucose measurements with an H202 based glucose electrode, in whole or diluted serum. Clinical measurements in whole serum are of great importance because of the need to avoid sample dilution and to save critical time in urgent situations. During measurements with a glucose electrode, the sample is not contaminated and not measurably consumed. This makes glucose analysis using an enzyme probe nondestructive and the same sample can be used for other analysis. Several different electrode membranes were studied, with the main interferences which can affect glucose measurements in whole or diluted serum. Several studies were carried out testing interferences at cathodic and anodic probes, both at constant potential and by linear sweep voltammetry. 114
GLUCOSE
MTHODS
PROBE INTERFERENCES
AND MATERIALS
Apparatus. A Bioanalytical Systems Inc. CV- 1A Voltammetry Control System and a Tacussel PRG-DEL electrochemical HPLC detector were used for constant potential studies performed anodically at +650 mV, as well as cathodically at -200 and -620 mV. The signals were measured with a Fluke Model 8050A digital voltmeter and a Houston Instruments Omniscribe strip chart recorder. The glucose electrode used was a Universal Sensors 2000-l glucose electrode covered with a glucose oxidase membrane (Universal Sensors, Inc.) and a 3500 molecular weight cutoff dialysis membrane. A Tacussel PRGE-DEC polarograph unit was used for linear sweep voltammetric studies, performed at +630 mV. Signals were monitored with the panel meter, a Lazar digital pH meter, and a Houston Instruments Omniscribe strip chart recorder. The electrode used for linear sweep measurements was a Universal Sensors 2000- 1 glucose electrode with no membrane, or with various membranes as described. Materials and chemicals. The dialysis membranes were 3500 molecular weight cutoff (MWCO),* Spectra/Par 3, from Spectrum Medical Industries Inc., Los Angeles, California. The glucose enzyme membranes were from Universal Sensors Inc., New Orleans, Louisiana. The 100 MWCO membranes were prepared according to the procedure of Taylor and others (4). Nafion coatings were prepared from a 5.0 wt% solution of the polymer from Solution Technology, Mendenhall, Pennsylvania. Nafion membranes were formed by dipping the electrode into the Nanon solution and letting the wet layer air-dry. This was repeated two additional times. The buffer used was a physiological phosphate buffer, pH 7.4, consisting of 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HP04, and 1.5 mM KH2P04. Ascorbic acid, uric acid, glutathione, acetaminophen, and cysteine * Abbreviation used: MWCO, molecular weight cut-off.
115
IN BLOOD GLUCOSE
were obtained from Sigma Chemical Company. All other chemicals were reagent grade. Ten serum samples (collected without anticoagulant) were obtained from Touro Infirmary, New Orleans, Louisiana. Stock solutions of interferants were prepared, at concentrations of 5 and 10 mg/dl, and also solid amounts were mixed with whole blood or serum to known levels. If interferences were noted at 10 mg/dl, higher levels were prepared, as required. PROCEDURES
Constant Potential Studies Glucose response curves were obtained in the absence and presence of the interferences: ascorbic acid, uric acid, cysteine, glutathione, aspirin, and acetaminophen. The levels of interferences were 5 and 10 mg/dl except for uric acid which was 6.9 mg/dl. Measurements were made at +650, -200, and -620 mV vs an Ag/AgCl reference electrode. All currents were measured in quiescent solution after 3 min, when over 90% of the response is obtained with all of the dierent membranes tested. Glucose calibration curves at applied potentials of +650, -200, and -620 mV were recorded for membranes consisting of an enzyme and a 3500 MWCO membrane. Measurements at -620 mV were very erratic and unreliable. Those at -200 mV were better; however, only those measurements at +650 were considered reliable. Linear Sweep Voltammetric
Studies
Several linear sweep voltammetric studies were performed at a scan rate of 10 mV/s using the Universal Sensors electrode. These included studies using: (1) The 3500 MWCO membrane in whole and diluted sera, and (2) The 3500 MWCO + glucose + 100 MWCO membranes in: (a) diluted and whole sera, (b) diluted and whole serum spiked with
116
PALLESCHI
interferences, and (c) diluted and whole serum spiked with glucose. The 3500 MWCO membrane was chosen, since it represents the dialysis material used most commonly in clinical assays. The 100 MWCO is used in many clinical instruments, e.g., the Yellow Springs analyzers and the Fuji Electronic analyzer (Tokyo), to remove electroactive compounds. pH Studies Linear sweep voltammograms were taken of hydrogen peroxide and the interferences, in buffer at pH 6.0, 7.5, and 8.5. The electrode used was a Universal Sensors Glucose electrode covered with a 3500 MWCO membrane and no enzyme. The reference electrode was Ag/AgCl and the reference solution was the physiological phosphate buffer. The linear sweep was from 0.0 to 0.9 V at a rate of 10 mV/s. The electrode was pretreated in the appropriate buffer before each sweep by applying -0.20 V for 3 min, then 0.00 V for 1 min. The solutions were not stirred. The physiological phosphate buffer was prepared as usual and was titrated with phosphoric acid to pH 6.0, or with a sodium hydroxide solution to pH 8.5. RESULTS AND DISCUSSION
Constant Potential Studies Typical glucose calibration curves at +650 mV demonstrated that, in general, the glucose response of an electrode with an enzyme membrane and a 3500 MWCO membrane was decreased by approximately 20% by the addition of a 100 MWCO membrane and by 70% by the addition of a Nafion membrane. The curves obtained at +650 mV with and without various levels of the interferences, are given in Fig. 1. Variations in the no interference curve A are due to differences in the base electrodes and the prepared membranes used. Comparison should be made between A, and B, and C, in each case. With only the 3500 MWCO membrane the most serious interfer-
ET AL.
ence was cysteine, followed by acetaminophen, ascorbic acid, and gluthathione. Although Nafion decreased the glucose response by 70%, it did not eliminate, or selectively reduce, any of the interferences, except possibly ascorbic acid. It is not recommended for practical use. The 100 MWCO membrane eliminated all interferences, except acetaminophen, and is recommended for use. As little as 0.01 mg/dl of glucose can be measured, the linear range extends to 200 mg/dl, and the precision at normal levels of glucose (So-120 mgjdl) is about 2%. pH Studies It has been previously demonstrated (3) that the half wave potential of hydrogen peroxide decreases with increasing pH at a rate of approximately 60 mV/pH unit. The effect of pH on the half wave potentials of the interferences was studied to determine if any interferences, acetaminophen in particular, could be reduced or eliminated by sample pH adjustment. Unfortunately the voltammograms of acetaminophen and hydrogen peroxide shift with pH changes, similar to H202. The other interferences were hard to evaluate because their responses were low and the residual currents of the three buffers also shifted with the pH. pH Adjustment is, thus, not a feasible means of reducing the interferences. Linear Sweep Voltammetric
Studies
All interferences were studied on both anodic and cathodic scans with one of the following membranes: (1) no membrane, (2) a glucose membrane and 3500 MWCO membrane and (3) a glucose membrane, 3500 MWCO membrane, and a 100 MWCO membrane with the 100 MWCO membrane next to the electrode. Anodic linear sweep voltammagrams are given in Fig. 2 for the case with no membrane (bare platinum). The voltammograms of the buffer and compounds vary, depending on the electrode pretreatment and history. Table 1
GLUCOSE 3500
ACET
PROBE
INTERFERENCES 100
YWCO
ASPIRIN
GLUTATHIONE
ACID
BLOOD
j;:,-l
i..-
m
~
117
GLUCOSE
MWCO
NAFtON
~
AMt NOP “ENH
ASCORBIC
IN
m
~~~
+-----$-,a, 0
.
.
100
100
GLUCOSE
CONCENTRATION
tms 4
Ftc. 1. Response curves in the presence and absence of interferences with various membranes. (A) No interference present, (B) 5 mg/dl interference present (in case of uric acid, 6.9 mg/dl was used), (C) 10 mg/ dl interference present.
200
118
PALLESCHI
ET AL.
Measurements of Diluted and Whole Serum with the 3500 MWCO Membrane Measuring in whole or diluted serum we always use the 3500 MWCO (dialysis) membrane to protect the probe from proteins which may be absorbed on the electrode surface or can interfere with the enzymatic membrane. All measurements were made pretreating the electrode in buffer for 3 min at -200 mV, then 1 min at 0 mV before scanning. Ten sera 0 300 600 900 samples diluted l/ 10 with buffer and ten whole POTENTIAL. m” sera samples were measured immediately after FIG. 2. Typical anodic voItammograms at a bare platinum electrode. 0 = Buffer, glutathion, and aspirin (10 the electrode pretreatment. Diluted and whole after the elecmg/dl), 1 = Cysteine (10 mgjdl), 2 = HzOz (17 m&B), 3 sera were scanned immediately trode was dipped into the test solution to sim= Acetaminophen (10 mg/dl), 4 = Uric acid (6.7 mgJd1, saturated solution), and 5 = Ascorbic acid (2.5 mg/dl). ulate an in vivo measurement. No large differences between diluted blood samples were observed measuring in this way. At +630 mV gives the half wave potentials for the anodic the average current change between buffer and oxidation waves, and Table 2 gives the current diluted sera was approximately 40 nA. For change from that of the buffer for various levels buffer and whole serum it was 125 nA. The average buffer background current at 630 mV of the interferences in buffer at 630 mV. was 630 nA. pH Measurements were made When scanning anodically, with no membefore and after scans. The pH was 8.5 + 0.1 brane, a significant signal was observed from all substances tested except glutathione and for whole sera and 7.7 f 0.2 for diluted sera. aspirin. Cysteine gave the highest response, After 3-5 h at room temperature a variation of 0.1 pH unit in whole and diluted sera was then acetaminophen. When any of the membranes were used, only acetaminophen and observed. No substantial difference was observed hydrogen peroxide gave significant responses. Cathodically, only hydrogen peroxide gave a when measuring whole sera immediately after significant response. There was only a very electrode pretreatment or 10 min after presmall response to cysteine and glutathione. Considering the anodic half wave potentials, TABLE 1 most interferences can be reduced by judicious ANODIC HALF WAVE POTENTIALS OF selection of the applied potential. This is espeSOME INTERFERENCES cially beneficial for the reduction of the most Substance Half wave potential (mV) serious interference, acetaminophen. The linear sweep voltammetric response to 150 Hz02 glucose was measured in quiescent solutions Ascorbic acid 50 with the two enzyme membrane systems, 3500 Uric acid 330 MWCO + enzyme and 3500 MWCO + enCysteine 330 * Glutathione zyme + 100 MWCO. The addition of the 100 Acetaminophen 400 MWCO membrane decreased the response to * Aspirin glucose at +630 mV by approximately 30%. This agrees with the earlier constant potential * Not determined because they did not give a measurstudies. able signal at 10 mg/dl between 0 and 900 mV.
GLUCOSE
PROBE INTERFERENCES
IN BLOOD
119
GLUCOSE
TABLE 2 CURRENT
AT
+630
DUE
TO VARIOUS
SUBSTANCES
AT A PLATINUM
ELECTRODE
WITH
DIFFERENT
MEMBRANES
Current change (nA) Substance Buffer Ascorbic acid
Concentration mg/dl
No membrane
Dialysis + enzyme
Dialysis + enzyme 100 MWCO
0.4 2.5 25.0
550 0 15 975
450 0 50
225 0 -
5.0 6.7
150 175
-
-
5.0 10.0 25.0
300 625 1750
-
Glutathione
10.0
Aspirin
10.0
Acetaminophen Hydrogen peroxide
Uric acid Cysteine
0
0 -
0 0
0 0
0
0
0
0
0
0
10.0
175
150
50
3.4 17.0 34.0
180 900 1700
75 200
100 200
Note. Except for the buffer currents, all currents are the total current minus the buffer current. Currents given as 0 are less than that detectable with the system used (i.e., ~25 nA).
treatment. We prefer the immediate measurement to avoid possible reference electrode contamination. The electrode was always washed with buffer to avoid protein precipitation on the dialysis membrane and to keep the same osmotic pressure between internal and external solutions. Measurements of Diluted and Whole Sera with 3500 MWCO + Glucose + 100 A4 WC0 Membranes During these studies we used a glucose membrane to better test the possible enzymatic interferences on the glucose electrode. The buffer’s background current was reduced by approximately 50% (300 nA at +630 mV) compared to an electrode with only a 3500 MWCO dialysis membrane. Figure 3 shows 10 curves each of buffer, I/ 10 diluted sera, and whole set-a.The measurements were made
in the same way as those described above. Curves of diluted sera were very close together and only a little different from the buffer curves (40 nA average difference). The curves of whole sera were quite different, possibly because of endogeneous glucose variations in the different sera. The average current change from the background buffer was 230 nA at 630 mV, which was 100 nA higher than that with a similar electrode without the enzyme membrane. Pretreating the glucose electrode electrochemically, as above, improved reproducibility.and is recommended. Measurements of Sera Samples Spiked with Interferences In the previous buffer studies it was demonstrated that in an anodic scan the only serious interference was acetaminophen. We
PALLESCHI
ET AL.
or 200 mg/dl glucose, then diluted. A good response to added glucose was observed. This was not observed scanning whole serum with added glucose. This was probably because of the endogeneous glucose level and nonlinear response of the electrode above 200 mg/dl, due to insufficient oxygen. CONCLUSION
With only a large molecular weight cut-off membrane, such as a 3500 MWCO dialysis, there are significant interferences from many potential blood constituents, including acetaminophen, cysteine, and ascorbic acid. Since the glucose enzyme reaction product detected is hydrogen peroxide, which has a molecular weight of 34, the best membrane configuration 600
300 POTENTIAL,
600
mV
FIG. 3. Anodic scan family of curves obtained using a electrode including a 100 MWCO membrane. 10 individual samples each OE A = Buffer, B = Diluted sera, l/10, and C = Whole sera.
ghcose
also studied the interferences, acetaminophen, cysteine, and ascorbic acid, in 1/ 10 diluted sera and in whole sera by adding known amounts of the solid interferants to such samples. Figure 4 is a typical set of curves with one of these interferences, acetaminophen, which at a concentration of 10 mg/dl gave an increase in current of 80 nA in diluted serum. In whole serum the increases in current were 40 and 70 nA, respectively, for acetaminophen present at 10 and 20 mg/dl. The increase in current was calculated versus diluted or whole serum without interference. No interference was observed in ascorbic acid or glutathione measurements. A very slight positive interference was observed with 10 mg/dl cysteine in diluted serum. No interference was observed with cysteine at 10 mg/dl in whole serum. Studies Adding Glucose in Whole and Diluted Sera Figure 5 shows different curves of pooled, diluted sera and pooled serum spiked with 100
0
300 POTENTIAL,
600 mV
900
FIG. 4. Effect of acetaminophen on anodic scans obtained with a Glucose Electrode including a 100 MWCO membrane. A = Buffer, B = Diluted serum, l/10, C = Diluted serum, l/ 10 plus acetaminophen (IO mg/dl), and D = Whole serum. E = Whole serum plus acetaminophen (10 mgjdl). F = Whole serum plus acetaminophen (20 mg/dl).
GLUCOSE
PROBE INTERFERENCES
IN BLOOD GLUCOSE
121
to eliminate electrochemical interferences should incorporate a 50 to 100 molecular weight cut-off membrane next to the platinum electrode. Using such a membrane, the only serious interference was acetaminophen, the active ingredient in Tylenol or Advil. The 100 MWCO membrane as prepared by the reference procedure passed substances of molecular weight slightly greater than 100. Constructing a glucose electrode with a 100 MWCO internal membrane, glucose oxidase and a 3500 MW (dialysis) outer membrane, from 0.01 to 200 mg/dl glucose can be determined with a precision at 100 mg/dl of +2%. ACKNOWLEDGMENT We acknowledge financial assistance from Liston Scientific Company, Newport Beach, California. REFERENCES 800 POTENTIAL,
mV
FIG. 5. Anodic scan family of curves obtained using a Glucose Electrode including a 100 MWCO membrane. A = Buffer, B = Diluted serum, l/10, C = Diluted serum, l/IO plus glucose, 100 mg/dl, and D = Diluted serum, l/10 plus glucose, 200 mg/dl.
I. Hicks, G. P., and Updike, S. J. U.S. Patent 1970: 3,542,662, Nov. 24. 2. Nanjo, M., and Guilbault, G. G. (1974) Anal. Chim. Acta 73, 367-373.
3. Guilbault, G. G., and Lubrano. G. J. (1973) Anal. Chim.
Acta 64, 439.
4. Taylor, P. S., Kmetec, E., and Johnson, J. M. (1977) Anal. Chem.
49,789-794.
BKXIHEMISTRY
13, I 14-12 1 ( 1986)
A Study of Interferences in Glucose Measurements in Blood by Hydrogen Peroxide Based Glucose Probes GIUSEPPEPALLESCHI,~.' MOHAMMADALINABIRAHNI,-~GLENN J. NGEH NGWAINBI,~ANDGEORGEG.GUILBAULT*~~
J. LUBRANO,*
*Universal Sensors, Inc., 5258 Veterans Memorial Boulevard, Metairie, Louisiana 70006; and tDepartment of Chemistry, University of New Orleans, New Orleans, Louisiana 70148 Received April 24. I986 The main blood constituents which could interfere in clinical glucose measurements using a hydrogen peroxide based glucose electrode have been investigated using severaldifferent membranes and constant and sweeping potentials. Roth diluted and whole undiluted sera were investigated. With a 3500 molecular weight cut-off (MWCO) membrane, acetaminophen, cysteine, and ascorbic acid can interfere. With a 100 MWCO membrane, only acetaminophen interfered. Q 1986academic Press, Inc. KEY
WORDS: glucose electrode; interferences; vohammetry; membranes.
The importance of glucose determinations for the diagnosis and treatment of diabetes has been well recognized. Before the twentieth century, over 30 methods for the measurement of glucose in biological fluids were already proposed. The desire for simplicity, precision, and good specificity has brought about the development of several hundred methods and modifications within the past several decades alone. The current methods used to maintain a normal glucose level in treating diabetes require daily, weekly, or monthly analysis of serum or urine. The most reliable electrochemical system for the assay of glucose utilizes an enzyme system, glucose oxidase (EC 1.1.3.4) according to the reaction: /3-D(+)glucose + 02 + Hz0 ‘“-zdase Hz02 + gluconic acid An assay of the glucose level can be made electrochemically either by monitoring the oxygen consumption with a platinum cathode ’ Present address: Department of Science and Chemical Technologies, IInd University of Rome, Tor Vergata, Italy 00173. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproductton in any form reserved.
(1,2), or by monitoring the Hz02 produced with a platinum anode (3). With the O2 system one generally looks at a small rate of current change in the presence of a large current background. With the H202 system, one looks at a similar rate or current change but in the presence of a much smaller background. The purpose of this work was to investigate some of the blood constituents which could interfere in clinical glucose measurements with an H202 based glucose electrode, in whole or diluted serum. Clinical measurements in whole serum are of great importance because of the need to avoid sample dilution and to save critical time in urgent situations. During measurements with a glucose electrode, the sample is not contaminated and not measurably consumed. This makes glucose analysis using an enzyme probe nondestructive and the same sample can be used for other analysis. Several different electrode membranes were studied, with the main interferences which can affect glucose measurements in whole or diluted serum. Several studies were carried out testing interferences at cathodic and anodic probes, both at constant potential and by linear sweep voltammetry. 114
GLUCOSE
MTHODS
PROBE INTERFERENCES
AND MATERIALS
Apparatus. A Bioanalytical Systems Inc. CV- 1A Voltammetry Control System and a Tacussel PRG-DEL electrochemical HPLC detector were used for constant potential studies performed anodically at +650 mV, as well as cathodically at -200 and -620 mV. The signals were measured with a Fluke Model 8050A digital voltmeter and a Houston Instruments Omniscribe strip chart recorder. The glucose electrode used was a Universal Sensors 2000-l glucose electrode covered with a glucose oxidase membrane (Universal Sensors, Inc.) and a 3500 molecular weight cutoff dialysis membrane. A Tacussel PRGE-DEC polarograph unit was used for linear sweep voltammetric studies, performed at +630 mV. Signals were monitored with the panel meter, a Lazar digital pH meter, and a Houston Instruments Omniscribe strip chart recorder. The electrode used for linear sweep measurements was a Universal Sensors 2000- 1 glucose electrode with no membrane, or with various membranes as described. Materials and chemicals. The dialysis membranes were 3500 molecular weight cutoff (MWCO),* Spectra/Par 3, from Spectrum Medical Industries Inc., Los Angeles, California. The glucose enzyme membranes were from Universal Sensors Inc., New Orleans, Louisiana. The 100 MWCO membranes were prepared according to the procedure of Taylor and others (4). Nafion coatings were prepared from a 5.0 wt% solution of the polymer from Solution Technology, Mendenhall, Pennsylvania. Nafion membranes were formed by dipping the electrode into the Nanon solution and letting the wet layer air-dry. This was repeated two additional times. The buffer used was a physiological phosphate buffer, pH 7.4, consisting of 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HP04, and 1.5 mM KH2P04. Ascorbic acid, uric acid, glutathione, acetaminophen, and cysteine * Abbreviation used: MWCO, molecular weight cut-off.
115
IN BLOOD GLUCOSE
were obtained from Sigma Chemical Company. All other chemicals were reagent grade. Ten serum samples (collected without anticoagulant) were obtained from Touro Infirmary, New Orleans, Louisiana. Stock solutions of interferants were prepared, at concentrations of 5 and 10 mg/dl, and also solid amounts were mixed with whole blood or serum to known levels. If interferences were noted at 10 mg/dl, higher levels were prepared, as required. PROCEDURES
Constant Potential Studies Glucose response curves were obtained in the absence and presence of the interferences: ascorbic acid, uric acid, cysteine, glutathione, aspirin, and acetaminophen. The levels of interferences were 5 and 10 mg/dl except for uric acid which was 6.9 mg/dl. Measurements were made at +650, -200, and -620 mV vs an Ag/AgCl reference electrode. All currents were measured in quiescent solution after 3 min, when over 90% of the response is obtained with all of the dierent membranes tested. Glucose calibration curves at applied potentials of +650, -200, and -620 mV were recorded for membranes consisting of an enzyme and a 3500 MWCO membrane. Measurements at -620 mV were very erratic and unreliable. Those at -200 mV were better; however, only those measurements at +650 were considered reliable. Linear Sweep Voltammetric
Studies
Several linear sweep voltammetric studies were performed at a scan rate of 10 mV/s using the Universal Sensors electrode. These included studies using: (1) The 3500 MWCO membrane in whole and diluted sera, and (2) The 3500 MWCO + glucose + 100 MWCO membranes in: (a) diluted and whole sera, (b) diluted and whole serum spiked with
116
PALLESCHI
interferences, and (c) diluted and whole serum spiked with glucose. The 3500 MWCO membrane was chosen, since it represents the dialysis material used most commonly in clinical assays. The 100 MWCO is used in many clinical instruments, e.g., the Yellow Springs analyzers and the Fuji Electronic analyzer (Tokyo), to remove electroactive compounds. pH Studies Linear sweep voltammograms were taken of hydrogen peroxide and the interferences, in buffer at pH 6.0, 7.5, and 8.5. The electrode used was a Universal Sensors Glucose electrode covered with a 3500 MWCO membrane and no enzyme. The reference electrode was Ag/AgCl and the reference solution was the physiological phosphate buffer. The linear sweep was from 0.0 to 0.9 V at a rate of 10 mV/s. The electrode was pretreated in the appropriate buffer before each sweep by applying -0.20 V for 3 min, then 0.00 V for 1 min. The solutions were not stirred. The physiological phosphate buffer was prepared as usual and was titrated with phosphoric acid to pH 6.0, or with a sodium hydroxide solution to pH 8.5. RESULTS AND DISCUSSION
Constant Potential Studies Typical glucose calibration curves at +650 mV demonstrated that, in general, the glucose response of an electrode with an enzyme membrane and a 3500 MWCO membrane was decreased by approximately 20% by the addition of a 100 MWCO membrane and by 70% by the addition of a Nafion membrane. The curves obtained at +650 mV with and without various levels of the interferences, are given in Fig. 1. Variations in the no interference curve A are due to differences in the base electrodes and the prepared membranes used. Comparison should be made between A, and B, and C, in each case. With only the 3500 MWCO membrane the most serious interfer-
ET AL.
ence was cysteine, followed by acetaminophen, ascorbic acid, and gluthathione. Although Nafion decreased the glucose response by 70%, it did not eliminate, or selectively reduce, any of the interferences, except possibly ascorbic acid. It is not recommended for practical use. The 100 MWCO membrane eliminated all interferences, except acetaminophen, and is recommended for use. As little as 0.01 mg/dl of glucose can be measured, the linear range extends to 200 mg/dl, and the precision at normal levels of glucose (So-120 mgjdl) is about 2%. pH Studies It has been previously demonstrated (3) that the half wave potential of hydrogen peroxide decreases with increasing pH at a rate of approximately 60 mV/pH unit. The effect of pH on the half wave potentials of the interferences was studied to determine if any interferences, acetaminophen in particular, could be reduced or eliminated by sample pH adjustment. Unfortunately the voltammograms of acetaminophen and hydrogen peroxide shift with pH changes, similar to H202. The other interferences were hard to evaluate because their responses were low and the residual currents of the three buffers also shifted with the pH. pH Adjustment is, thus, not a feasible means of reducing the interferences. Linear Sweep Voltammetric
Studies
All interferences were studied on both anodic and cathodic scans with one of the following membranes: (1) no membrane, (2) a glucose membrane and 3500 MWCO membrane and (3) a glucose membrane, 3500 MWCO membrane, and a 100 MWCO membrane with the 100 MWCO membrane next to the electrode. Anodic linear sweep voltammagrams are given in Fig. 2 for the case with no membrane (bare platinum). The voltammograms of the buffer and compounds vary, depending on the electrode pretreatment and history. Table 1
GLUCOSE 3500
ACET
PROBE
INTERFERENCES 100
YWCO
ASPIRIN
GLUTATHIONE
ACID
BLOOD
j;:,-l
i..-
m
~
117
GLUCOSE
MWCO
NAFtON
~
AMt NOP “ENH
ASCORBIC
IN
m
~~~
+-----$-,a, 0
.
.
100
100
GLUCOSE
CONCENTRATION
tms 4
Ftc. 1. Response curves in the presence and absence of interferences with various membranes. (A) No interference present, (B) 5 mg/dl interference present (in case of uric acid, 6.9 mg/dl was used), (C) 10 mg/ dl interference present.
200
118
PALLESCHI
ET AL.
Measurements of Diluted and Whole Serum with the 3500 MWCO Membrane Measuring in whole or diluted serum we always use the 3500 MWCO (dialysis) membrane to protect the probe from proteins which may be absorbed on the electrode surface or can interfere with the enzymatic membrane. All measurements were made pretreating the electrode in buffer for 3 min at -200 mV, then 1 min at 0 mV before scanning. Ten sera 0 300 600 900 samples diluted l/ 10 with buffer and ten whole POTENTIAL. m” sera samples were measured immediately after FIG. 2. Typical anodic voItammograms at a bare platinum electrode. 0 = Buffer, glutathion, and aspirin (10 the electrode pretreatment. Diluted and whole after the elecmg/dl), 1 = Cysteine (10 mgjdl), 2 = HzOz (17 m&B), 3 sera were scanned immediately trode was dipped into the test solution to sim= Acetaminophen (10 mg/dl), 4 = Uric acid (6.7 mgJd1, saturated solution), and 5 = Ascorbic acid (2.5 mg/dl). ulate an in vivo measurement. No large differences between diluted blood samples were observed measuring in this way. At +630 mV gives the half wave potentials for the anodic the average current change between buffer and oxidation waves, and Table 2 gives the current diluted sera was approximately 40 nA. For change from that of the buffer for various levels buffer and whole serum it was 125 nA. The average buffer background current at 630 mV of the interferences in buffer at 630 mV. was 630 nA. pH Measurements were made When scanning anodically, with no membefore and after scans. The pH was 8.5 + 0.1 brane, a significant signal was observed from all substances tested except glutathione and for whole sera and 7.7 f 0.2 for diluted sera. aspirin. Cysteine gave the highest response, After 3-5 h at room temperature a variation of 0.1 pH unit in whole and diluted sera was then acetaminophen. When any of the membranes were used, only acetaminophen and observed. No substantial difference was observed hydrogen peroxide gave significant responses. Cathodically, only hydrogen peroxide gave a when measuring whole sera immediately after significant response. There was only a very electrode pretreatment or 10 min after presmall response to cysteine and glutathione. Considering the anodic half wave potentials, TABLE 1 most interferences can be reduced by judicious ANODIC HALF WAVE POTENTIALS OF selection of the applied potential. This is espeSOME INTERFERENCES cially beneficial for the reduction of the most Substance Half wave potential (mV) serious interference, acetaminophen. The linear sweep voltammetric response to 150 Hz02 glucose was measured in quiescent solutions Ascorbic acid 50 with the two enzyme membrane systems, 3500 Uric acid 330 MWCO + enzyme and 3500 MWCO + enCysteine 330 * Glutathione zyme + 100 MWCO. The addition of the 100 Acetaminophen 400 MWCO membrane decreased the response to * Aspirin glucose at +630 mV by approximately 30%. This agrees with the earlier constant potential * Not determined because they did not give a measurstudies. able signal at 10 mg/dl between 0 and 900 mV.
GLUCOSE
PROBE INTERFERENCES
IN BLOOD
119
GLUCOSE
TABLE 2 CURRENT
AT
+630
DUE
TO VARIOUS
SUBSTANCES
AT A PLATINUM
ELECTRODE
WITH
DIFFERENT
MEMBRANES
Current change (nA) Substance Buffer Ascorbic acid
Concentration mg/dl
No membrane
Dialysis + enzyme
Dialysis + enzyme 100 MWCO
0.4 2.5 25.0
550 0 15 975
450 0 50
225 0 -
5.0 6.7
150 175
-
-
5.0 10.0 25.0
300 625 1750
-
Glutathione
10.0
Aspirin
10.0
Acetaminophen Hydrogen peroxide
Uric acid Cysteine
0
0 -
0 0
0 0
0
0
0
0
0
0
10.0
175
150
50
3.4 17.0 34.0
180 900 1700
75 200
100 200
Note. Except for the buffer currents, all currents are the total current minus the buffer current. Currents given as 0 are less than that detectable with the system used (i.e., ~25 nA).
treatment. We prefer the immediate measurement to avoid possible reference electrode contamination. The electrode was always washed with buffer to avoid protein precipitation on the dialysis membrane and to keep the same osmotic pressure between internal and external solutions. Measurements of Diluted and Whole Sera with 3500 MWCO + Glucose + 100 A4 WC0 Membranes During these studies we used a glucose membrane to better test the possible enzymatic interferences on the glucose electrode. The buffer’s background current was reduced by approximately 50% (300 nA at +630 mV) compared to an electrode with only a 3500 MWCO dialysis membrane. Figure 3 shows 10 curves each of buffer, I/ 10 diluted sera, and whole set-a.The measurements were made
in the same way as those described above. Curves of diluted sera were very close together and only a little different from the buffer curves (40 nA average difference). The curves of whole sera were quite different, possibly because of endogeneous glucose variations in the different sera. The average current change from the background buffer was 230 nA at 630 mV, which was 100 nA higher than that with a similar electrode without the enzyme membrane. Pretreating the glucose electrode electrochemically, as above, improved reproducibility.and is recommended. Measurements of Sera Samples Spiked with Interferences In the previous buffer studies it was demonstrated that in an anodic scan the only serious interference was acetaminophen. We
PALLESCHI
ET AL.
or 200 mg/dl glucose, then diluted. A good response to added glucose was observed. This was not observed scanning whole serum with added glucose. This was probably because of the endogeneous glucose level and nonlinear response of the electrode above 200 mg/dl, due to insufficient oxygen. CONCLUSION
With only a large molecular weight cut-off membrane, such as a 3500 MWCO dialysis, there are significant interferences from many potential blood constituents, including acetaminophen, cysteine, and ascorbic acid. Since the glucose enzyme reaction product detected is hydrogen peroxide, which has a molecular weight of 34, the best membrane configuration 600
300 POTENTIAL,
600
mV
FIG. 3. Anodic scan family of curves obtained using a electrode including a 100 MWCO membrane. 10 individual samples each OE A = Buffer, B = Diluted sera, l/10, and C = Whole sera.
ghcose
also studied the interferences, acetaminophen, cysteine, and ascorbic acid, in 1/ 10 diluted sera and in whole sera by adding known amounts of the solid interferants to such samples. Figure 4 is a typical set of curves with one of these interferences, acetaminophen, which at a concentration of 10 mg/dl gave an increase in current of 80 nA in diluted serum. In whole serum the increases in current were 40 and 70 nA, respectively, for acetaminophen present at 10 and 20 mg/dl. The increase in current was calculated versus diluted or whole serum without interference. No interference was observed in ascorbic acid or glutathione measurements. A very slight positive interference was observed with 10 mg/dl cysteine in diluted serum. No interference was observed with cysteine at 10 mg/dl in whole serum. Studies Adding Glucose in Whole and Diluted Sera Figure 5 shows different curves of pooled, diluted sera and pooled serum spiked with 100
0
300 POTENTIAL,
600 mV
900
FIG. 4. Effect of acetaminophen on anodic scans obtained with a Glucose Electrode including a 100 MWCO membrane. A = Buffer, B = Diluted serum, l/10, C = Diluted serum, l/ 10 plus acetaminophen (IO mg/dl), and D = Whole serum. E = Whole serum plus acetaminophen (10 mgjdl). F = Whole serum plus acetaminophen (20 mg/dl).
GLUCOSE
PROBE INTERFERENCES
IN BLOOD GLUCOSE
121
to eliminate electrochemical interferences should incorporate a 50 to 100 molecular weight cut-off membrane next to the platinum electrode. Using such a membrane, the only serious interference was acetaminophen, the active ingredient in Tylenol or Advil. The 100 MWCO membrane as prepared by the reference procedure passed substances of molecular weight slightly greater than 100. Constructing a glucose electrode with a 100 MWCO internal membrane, glucose oxidase and a 3500 MW (dialysis) outer membrane, from 0.01 to 200 mg/dl glucose can be determined with a precision at 100 mg/dl of +2%. ACKNOWLEDGMENT We acknowledge financial assistance from Liston Scientific Company, Newport Beach, California. REFERENCES 800 POTENTIAL,
mV
FIG. 5. Anodic scan family of curves obtained using a Glucose Electrode including a 100 MWCO membrane. A = Buffer, B = Diluted serum, l/10, C = Diluted serum, l/IO plus glucose, 100 mg/dl, and D = Diluted serum, l/10 plus glucose, 200 mg/dl.
I. Hicks, G. P., and Updike, S. J. U.S. Patent 1970: 3,542,662, Nov. 24. 2. Nanjo, M., and Guilbault, G. G. (1974) Anal. Chim. Acta 73, 367-373.
3. Guilbault, G. G., and Lubrano. G. J. (1973) Anal. Chim.
Acta 64, 439.
4. Taylor, P. S., Kmetec, E., and Johnson, J. M. (1977) Anal. Chem.
49,789-794.