Anal. Chem. 1998, 70, 2156-2162
Amperometric Microcell for Enzyme Activity Measurements Geza Nagy, Clarke X. Xu, and Richard P. Buck*
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290 Erno Lindner
Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708-0295 Michael R. Neuman
Department of Obstetrics and Gynecology, Metro Health Medical Center, Case Western Reserve University, Cleveland, Ohio 44109-1998
A small-volume cell was developed for enzyme activity measurements. The cell contained a detector chip, which was prepared using photolithographic and electrochemical techniques. The enzyme-catalyzed reaction takes place in a thin film inside the cell, and the concentration changes are followed in time with an amperometric detector. The combination of the thin-layer reaction medium and surface detection allows achievement of high sensitivity with very low sample volumes. The cell can be prepared as an inexpensive single-shot test device. The analytical performance of the cell was checked with several enzymes of clinical importance. Demonstration measurements were made with robust glucose oxidase. Then, an analytical procedure was worked out for the measurement of putrescine oxidase activity, an indicator for premature rupture of the amniotic membrane. Finally, two different reaction schemes were investigated for the activity determination of the myocardial infarction marker, creatine kinase. Owing to its simplicity and high sensitivity, amperometry is one of the most often used electroanalytical methods. Amperometric enzyme-based sensors are well-known and are often used both in research and in clinical analyzers. Their function is based on selective, enzyme-catalyzed chemical reactions proceeding in one or more enzyme-loaded reaction layers attached to the measuring surface of an amperometric sensing element of high sensitivity. Recently, a flat-form amperometric microsensor chip was developed in our laboratory using photolithographic and electrochemical technology.1-5 It has proven to be a good platform for biosensors.4,6 In the work reported here, a modified version of * To whom correspondence should be addressed. (1) Lindner, E.; Cosofret, V. V.; Ufer, S.; Kusy, R. P.; Buck, R. P.; Ash, R. B.; Nagle, H. T. J. Chem. Soc., Faraday Trans. 1, 1993, 89, 361-367. (2) Cosofret, V. V.; Erdosy, M.; Johnson, T. A.; Buck, R. P.; Ash, R. B.; Neuman, M. R. Anal. Chem. 1995, 67, 1647-1653. (3) Madaras, M. B.; Popescu, I. C.; Ufer, S.; Buck, R. P. Anal. Chim. Acta 1996, 319, 335-345. (4) Madaras, M. B.; Buck, R. P. Anal. Chem. 1996, 68, 3832-3839.
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that amperometric sensor chip was used for enzyme activity measurements; the applicability of this modified cell design was investigated. Glucose oxidase, creatine kinase, and putrescine oxidase enzyme activity responses of the cell were studied. The hydrogen peroxide formed in the enzyme-catalyzed reactions was amperometrically detected. The signal of an enzyme-based sensor depends on the enzyme activity in the reaction layer. By replacing the constant enzyme activity reaction layer of an enzyme electrode with the sample solution film and using constant substrate concentrations, enzyme activity determinations can be made. However, in the past, this approach could only be used in cases of high enzyme activity samples.7,8 In most enzyme activity measurements, the kinetics of the enzyme-catalyzed reactions are followed under optimal conditions (temperature, pH, and substrate concentration, etc.). The concentration change of a reaction partner or product is measured in the bulk of the media after the sample of unknown enzyme activity is introduced. Spectrophotometry, a bulk concentration method, is most often the method of choice for this “kinetic” type of enzyme activity measurement. We will show that the combination of an amperometric surface sensor and a thin-film reaction layer will result in a similarly harmonious match with respect to sensitivity. In our application, the high sensitivity is consistent with small sample size and minimal reagent consumption. EXPERIMENTAL SECTION Preparation of the Electrodes. Photolithographically fabricated, individually accessible gold plate twin electrodes with surface areas of 3.0 mm2 on flexible polyimide film (Kapton, DuPont) of 125 µm thickness were used for the preparation of the amperometric working and reference electrodes of the cell. In the present design, each 3-in. × 3-in. wafer contains 27 amperometric microcells (3 rows of 9). The description of the (5) Buck, R. P.; Cosofret, V. V.; Lindner, E.; Ufer, S.; Madaras, M. B.; Johnson, T. A.; Ash, R. B.; Neuman, M. R. Electroanalysis 1995, 9, 846-851. (6) Xu, C. X.; Marzouk, S. A. M.; Cosofret, V. V.; Buck, R. P.; Neuman, M. R.; Sprinkle, R. H. Talanta 1997, 44, 1625-1632. (7) Montalvo, J. G., Jr. Anal. Chem. 1969, 41, 2093. (8) Montalvo, J. G., Jr. Anal. Biochem. 1970, 38, 357. S0003-2700(97)01221-3 CCC: $15.00
© 1998 American Chemical Society Published on Web 04/02/1998
fabrication procedure was given earlier.9 First, a single amperometric cell with its bonding pads was cut from the wafer, and the connecting wires were attached to the bonding pads with silver epoxy (Epoxy Technology, Inc.). Next, the bonding pads were insulated with another epoxy layer (Quik-Stix, GC Electronics). Finally, one of the gold plate electrodes was electroplated with a shiny platinum layer, while the other was electrochemically converted to a Ag/AgCl reference electrode.10,11 To provide size exclusion-based permselectivity for amperometric hydrogen peroxide detection, an electrochemically formed, thin poly(m-phenylenediamine) (poly-mPDA) film was deposited on the platinum working electrode surface as worked out and reported.12 Generally, five or six electrodes were prepared at the same time. The electrodes were placed in 0.01 M phenylenediamine solution in 0.1 M phosphate buffer of pH 7.2. Cyclic voltammetric scans were made in the range of +0.2-0.8 V vs Ag/ AgCl reference electrode with 2 mV/s scan rate. Finally, the electrodes were individually checked. They were accepted for further use if they detected hydrogen peroxide at 0.6 V but showed less than 5 nA current increase when challenged with 0.2 mM ascorbic acid.13,14 The failure rate was less than 10%. Fabrication of the Thin-Layer Measuring Cell. The polyimide (Kapton) film holding the two-electrode cell, the insulating lead wires, and the bonding pads was attached to a supporting plate for easier handling. A flat surface of any solid material is appropriate. Microscope slides or special ceramic plates (Laser Tech, Minnetonka, MN) were used in this work. A thin insulating blacktape cover of about 200 µm thickness was stretched and pasted over the plate to hold down the polyimide film. A circular opening (with a diameter of 7 mm) was previously punched out of the blacktape, and then it was placed centrally over the original opening for the electrodes. In this way, the depth of the slightly recessed circular region was increased by the thickness of the blacktape. The small-volume space, called the reaction well, could hold a reagent-containing porous membrane disk or a liquid reagent film. To avoid solvent evaporation during measurements, a screw cap of a small vial or a plastic cover was used as reaction well top. Figure 1 shows the schematic design of the cell. The volume of the reaction well was about 20-25 µL. Preparation of Reagent Disks. The formation of a welldefined layer of reagent on the bottom of the reaction well is one of the key issues of this application. A hydrophilic, thin, porous matrix, employed at the surface, simplifies the film formation and improves the stability. Surface tension-decreasing additives, such as 1% hydroxypropyl cellulose, carboxymethyl cellulose, Triton, or Tween 20, and a film-holding window, placed in front of the sensing surface, helped further the formation of a homogeneous film. Well-functioning, single-shot “disposable” cells could be (9) Marzouk, S. A. M.; Xu, C. X.; Cosofret, B. R.; Buck, R. P.; Hassan, S. S. M.; Neuman, M. R.; Sprinkle, R. H. Anal. Chim. Acta, in press. (10) Blum, W.; Hogaboom, G. B. Principles of Electroplating and Electroforming, 3rd ed.; McGraw-Hill: New York, 1949; p 382. (11) Ives, D. J. G.; Janz, G. J. Reference Electrodes; Academic Press: New York, 1961. (12) Marzouk, S. A. M.; Cosofret, V. V.; Buck, R. P.; Yang, H.; Cascio, W. E.; Hassan, S. S. M. Talanta 1997, 44, 1527-1542. (13) Urban, G.; Jobst, G.; Aschauer, E.; Tilado, O.; Svasek, P.; Varahram, M. Sens. Actuators B 1994, 18-19, 592-596. (14) Moser, L.; Jobst, G.; Aschauer, E.; Svasek, P.; Varahram, M.; Urban, G.; Zanin, U. A.; Tjoutrina, G. Y.; Zharikova, A.; Berezov, T. T. Biosens. Bioelectron. 1995, 10, 527-532.
Figure 1. (a) Schematic design of an amperometric microcell for enzyme activity measurements. The microfabricated elctrochemical cell (on flexible polyimide Kapton with traces and bonding pads) is fixed on a ceramic substrate. The blacktape defines the reaction well that contains the porous disk and is covered with the cell top. (b) Schematic edge-on view of the cell. Electrode materials are shown.
obtained by experimenting with attached layers made of Kieselgel, cellulose acetate, and alumina powder using carboxymethyl cellulose as binding material. However, to make multiple measurements with the same cell, a replaceable matrix layer in the form of thin paper tissue disks (Whatman P81 filter paper, Kimwipes EX-L delicate task wipers, or Olympus lens cleaning tissues) were used in our experiments. Operation of the Cell. The working platinum and reference Ag/AgCl electrodes of the amperometric measuring chip, made on the surface of a Kapton film, are aligned in one plane. The layer structure of the cell is shown in Figure 1b. The porous filter paper guaranteed a uniform spreading of the small-volume reagent (analyte) and sample (enzyme solution). At the same time, it avoided droplet formation and convection. The small cap was used to avoid evaporation-related changes in the solution volumes. The cell is user-friendly; the fill-up with reagent, the introduction of the sample, and the cleaning do not need special care or skill. When a thin electrolyte film is coated on both electrode surfaces, Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
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amperometric measurements can be made, and changes of the concentration of an electroactive species can be detected. In our work, different enzyme-catalyzed reactions, resulting in hydrogen peroxide formation, were performed in thin buffered electrolyte film. Thus, the change of the current can indicate the rate of the enzymatic reaction proceeding in the electrolyte film. In the present examples, peroxide is the detected reaction product, and the amperometric current vs time curves were recorded. In the cases of glucose oxidase, and putrescine oxidase the enzymecatalyzed reaction between the substrate and the dissolved oxygen directly results in hydrogen peroxide formation. For creatine kinase, however, two further consecutive reaction steps led to H2O2 formation. High sensitivity, low reaction volumes, and sample sizes were accommodated. Procedure. Small volumes of buffered, substrate-containing reagent solutions were added into the amperometric sensor well. The current-time curves were recorded at a potential of 0.6 V vs Ag/AgCl reference electrode. After the current decreased to its low initial or background level, solution doses of known enzyme activity were added to the reagent cell. This simple two-electrode configuration was selected to avoid frequent problems with the three-electrode amperometric mode due to lost contact of the reference electrode. It can happen easily in a cell holding no more than a few microliters of electrolyte and can cause damage of the photolithographically prepared thinlayer working electrode unless a special potentiostat or guarding circuit is used. Chemicals. Creatine kinase (EC 2.7.3.2) enzyme prepared from rabbit muscle, creatine amidinohydrolase (creatinase, EC 3.5.3.3) from Actinobacillus species, adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate, phosphocreatine, 1,3-diaminobenzene, N-acetyl-L-cysteine, and glucose oxidase (EC 1.1.3.4) from Aspergillus niger were purchased from Sigma Chemical Co. (St. Louis, MO). Sarcosine oxidase (EC 1.5.3.1), glycerol kinase (glycerol 3-phosphotransferase, EC 2.7.1.30), L-R-glycerophosphate oxidase from a microorganism (EC 1.1.3.21), and putrescine oxidase (EC 1.4.3.10) from Micrococcus roseus were obtained from Toyobo Co., Ltd. Hydrogen hexachloroplatinate(IV) hydrate, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and hydroxypropyl cellulose were from Aldrich Chemical Co.; Merck (Rahway, NJ) magnesium acetate, Fisher Scientific Co. EDTA were used for preparing the solutions. All the solutions used in creatine kinase determinations were prepared with 100 mM HEPES buffer (pH 6.7) containing EDTA in 2.3 mM concentration to complex Ca2+ ions. Magnesium acetate at 11.5 mM activated the CK enzyme, and 0.7 g/L sodium chloride was used in the reference electrode. The measurements with putrescine oxidase and glucose oxidase were made in 100 mM pH 8.0 and pH 6.0 phosphate buffer, respectively, containing sodium chloride (7 g/L). The “standard” solutions with variable enzyme activities were always freshly prepared by serial dilution using chilled buffer solution of 0.1% bovine serum albumin (BSA) content. A small volume of stock solution (∼1 mg of enzyme in 100 mL of BSA-containing buffer) was diluted in several steps until the required activity range was reached. Thereafter, 50 mL of enzyme solution was mixed with different amounts of BSA containing buffer. The ready-to-use “standards” were kept on ice in microcentrifuge tubes between measurements. 2158 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
In most of the experiments, the enzyme activity values declared by the manufacturer were used. However, the creatine kinase and the putrescine oxidase enzyme activities were controlled time to time using the Sigma 45-1 Creatine Kinase Single Assays or the method described in the Toboyo Enzymes catalog (Toboyo Co., Osaka, Japan), respectively. In the latter procedure, the rate of hydrogen peroxide formation is measured at 510 nm spectrophotometrically:
2H2O2 + 4-aminoantipyrine + peroxidase
2,4-dichlorophenol 98 quinoneimine dye + 4H2O
RESULTS AND DISCUSSION Disks were punched out from different brands of paper tissue membranes and were used as the porous matrix. All three filter papers were appropriate in facilitating a homogeneous spreading of the reagent in the reaction well. The complete soaking of the Whatman paper was considerably slower than that of the other two. The Kimwipes EX-L wiper becomes completely soaked immediately, but it rolls up easily. The Olympus lens cleaning tissue keeps its contour, and it is fast in water uptake. Accordingly, most of the measurements used the latter material. However, practically any relatively thin, hydrophilic filter paper could be used. To carry out one measurement, the cell was opened and the reaction film supporting disk was placed on the sensing surface. A given volume (5-15 µL) of buffered substratecontaining reagent solution prepared with surface tension decreasing additive (e.g., with 1% hydroxypropyl cellulose) was added onto the disk to obtain an electrolyte film that was evenly spread and in contact with the electrodes. The cell was closed and connected to the measuring apparatus. The amperometric current was recorded continuously at 0.6 V working electrode potential. After obtaining a steady background current of 15-40 nA, a given volume of the enzyme-containing sample solution, typically a 10µL aliquot, was added and evenly distributed with the tip of a Hamilton syringe on the reaction film. Soon after, the amperometric current started to increase, indicating hydrogen peroxide production. The shapes of the current-time recordings showed similar characteristics for all three enzyme reactions studied but depended on the activity ranges of each enzyme. In the case of high enzyme activities, the current sharply increased initially and quickly achieved a sharp maximum. As the enzyme activity decreased, the maximum was shifted to the direction of longer time values and appeared less “pointy”. The current-time transients showed S-shaped, “polarographic step” curves when the enzyme activities were decreased further. However, in the range of practically important low enzyme activities, the current often continuously increases linearly. The shape of the current-time recording can be explained only qualitatively due the complicated nature of the enzyme reaction and amperometric detection in a stationary film. Local depletion of the substrate, or inhibition by the product, can be responsible for the maximum character. There may be a competition between anodic peroxide consumption and enzymatic peroxide production. A simple calculation shows that 10 nA current intensity oxidizes the hydrogen peroxide at the same rate that 3.1 µunits produces it in optimal conditions (considering that
Figure 2. Glucose oxidase response: current-time curves recorded after 2-µL aliquots of glucose oxidase solutions of different activities were added to 5 µL of 100 mM glucose solution on the disk: (a) 5.70, (b) 3.80, (c) 1.90, (d) 1.26, (e) 0.58, (f) 0.39, and (g) 0.19 unit/mL.
Figure 4. Putrescine oxidase calibration: current measured 200 s after injection (rate) vs PO enzyme activity. Ten microliters of 100 mM putrescine solution on the disk; 5 µL of enzyme solution added.
steady-state current. Poor reproducibility of initial wetting and the start time caused random measurement errors that were considerably larger when current values were measured at shorter times. On the other hand, the gain in precision or sensitivity was not substantial when current values measured at much longer times (e.g., 300 s) were used. In case of higher enzyme activities, peak-shaped response curves were obtained. The peak current intensities are also proportional to the enzyme activities. The putrescine oxidase (PO) catalyzes the following reaction: putrescine oxidase
H2N(CH2)4NH2 + O2 + H2O 98 H2N(CH2)3CHO + NH3 + H2O2 (1)
Figure 3. Glucose oxidase calibration: current measured at fixed time (200 s) vs enzyme activity. Five microliters of 100 mM glucose solution on the disk; 2-µL aliquots of glucose oxidase enzyme solution added.
1 unit of enzyme activity produces 1 µM hydrogen peroxide per minute). Measurements with One-Step Reactions. There are over 40 oxygen oxidoreductase enzymes which catalyze oxidations of different substrates by dissolved oxygen to produce hydrogen peroxide. The most often studied of them is glucose oxidase. Figure 2 shows amperometric current-time response curves recorded with the cell upon adding 5 µL of buffered glucose solution (100 mM, pH 6) and 2 µL of glucose oxidase enzyme solution of different activities onto the reaction membrane. Figure 3 is a glucose oxidase activity-response curve which was obtained by plotting the current values measured 200 s after initiation of the reaction against the activity of the enzyme solution. Each curve in Figure 2 (each point in Figure 3) was determined with a new filter paper and a new aliquot of substrate solution, i.e., the cell was disassembled, washed, and reassembled between measurements. The slopes of the current-time transients are also proportional to the enzyme activities. However, in this special case, there was no difference in the quality of the data (calibration curve or reproducibilities) when they were evaluated with the initial slope method, using the current data after 200 s, or after reaching the
The putrescine oxidase determinations are of importance in clinical diagnosis and in different areas of life science research. Amniotic fluid contains relatively high putrescine oxidase activity.15 It is believed that leaking of the amniotic fluid in case of premature rupture of the amniotic membrane can be detected by measuring the putrescine oxidase or diamine oxidase activity in the cervovaginal secretion.16-18 The putrescine oxidase response curves of the cell obtained with different enzyme activities are very similar to the curves shown in Figure 2. A putrescine oxidase calibration curve is shown in Figure 4, where current values, measured 200 s after enzyme addition, are plotted against the enzyme activity. A pH 8 Tris buffer was used as solvent, 10 µL of 100 mM putrescine solution was added on the filter paper disk, and 5-µL doses of putrescine oxidase enzyme solution were used to start the reaction. The current-time response curves recorded with the cell were sensitive to the total volume of the reaction media. To illustrate this, Figure 5 shows response curves made with different total (15) Tornquist, A.; Jonassen, F.; Johnson, P.; Fredholm, A. M. Acta Obstet. Gynecol. Scand. 1971, 50, 79-82. (16) Gahl, W. A.; Kozina, T. J.; Fuhrmann, D. D.; Vale, A. M. Obstet Gynecol. 1982, 60, 297-304. (17) Elmfors, B.; Tryding, N.; Tufvesson, G. J. Obstet. Gynecol. Br. Commonwealth 1974, 81, 361-362. (18) Bank, C. M.; Offermans, J. P.; Gijzen, A. H.; Smits, F.; van Dieijen-Visser, M. P.; Brombacher, P. Eur. J. Clin. Chem. Clin. Biochem. 1991, 29, 743748.
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Measurements with Creatine Kinase (CK). To investigate the applicability of the cell to enzymes other than oxygen oxidoreductases, creatine kinase activity determinations were selected. Creatine kinase is an intercellular enzyme. It catalyzes the re-phosphorylation process of ADP to ATP, thus recharging the energy pool of the cell. An increased level of creatine kinase activity in plasma indicates muscle or brain damage. The creatine kinase activity value is one of the myocardial heart attack markers. Most of the methods for CK activity measurements21 use the kinetically more favorable reverse reaction resulting in the hydrolysis of creatine phosphate in the presence of ADP. creatine kinase (pH 6.7)
Figure 5. Amperometric putrescine oxidase response curves recorded with different total solution volumes loaded to disk. The volume ratio of the substrate (100 mM putrescine) and enzyme solutions (6.6 units/mL) was always 2:1. Total volumes added to disk: (a) 4.5, (b) 6.0, (c) 7.5, (d) 9.0, (e) 10.5, (f) 15.0, (g) 18.0, and (h) 22.5 µL.
solution volumes but with the same ratio (2:1, v:v) of 100 mM putrescine solution and 6.6 units/mL enzyme solution. In the figure, each curve corresponds to a different total sample volume, split 2:1 between reagent and enzyme solution. Initial slopes of the response curves are nearly constant within a given range of total reaction volume (curves a-f). However, the steady-state currents of the response curves are nearly proportional to the solution volume in the reaction well. In relatively high volume media (curves g and h), the layer thickness becomes larger, and the transients become similar to the kinetic curves recorded with macroelectrodes in a large volume solution. Responses are less sensitive; slopes are reduced as compared with those from smaller sample volumes. More details on the putrescine oxidase response of the microcell and putrescine oxidase activity measurements are discussed elsewhere.19 Since sampling of the vaginal fluid for quantitative analytical measurements is quite complex, often the total enzyme activity on the sampling device (e.g., a stripe of filter paper) is determined.17,20 Diamine oxidase activities (DOA) in the range of 1-780 µunits were reported. Women with DOA >70 µunits had always ruptured membranes. If the amperometric microcell were used for the determination of putrescine oxidase in vaginal secretion, a sterile filter paper disk could serve both as a sampling device and as a hydrophilic porous matrix. Seventy microunits PO enzyme activity (in 11 µL total volume) gave well-developed signals in the amperometric microcell; i.e., the method is appropriate to detect premature rupture. However, we wish to emphasize that the main goal of this paper was to show the feasibility of the new method. Accordingly, we tried to test its general applicability instead of optimizing it for a single application. It is expected that the lower limit of detection can be extended to smaller enzyme activities by optimizing the cell, the procedures, reagent compositions, and volumes. This improvement, in combination with an appropriate sampling method, might be necessary since diamine oxidase activity in amniotic fluid (0.3-1.2 units/L) is below the smallest concentrations we tried to measure.18 (19) Nagy, G.; Xu, C. X.; Cosofret, V. V.; Buck, R. P.; Lindner, E.; Neuman, M. R.; Sprinkle, H., in preparation. (20) Wishart, M. M.; Jenkins, D. T.; Knott, M. L.; Aust, N. Z. J. Obstet. Gynecol., 1979, 19, 23-24.
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creatine phosphate + ADP 98 creatine + ATP (2) To use the amperometric cell, follow-up reactions generating hydrogen peroxide as one of the final products are needed. We can achieve this by further reacting either the creatine or the ATP. As the first example, liberated creatine can be hydrolyzed in a creatine amidinohydrolase (creatinase, EC 3.5.2.10) enzymecatalyzed reaction: creatinase
creatine + H2O 98 sarcosine + urea
(3)
As the third step, the sarcosine can be oxidized by the dissolved oxygen content of the medium under the catalytic action of sarcosine oxidase (EC 1.5.3.1): sarcosine oxidase
sarcosine + H2O + O2 98 glycine + HCHO + H2O2 (4) We call this reaction sequence one. Reaction steps 3 and 4 were successfully used in analytical procedures and in biosensors for creatinine and creatine determinations.2,22,23 If the ATP-based reaction pathway is considered, then the glycerol kinase (EC 2.7.1.30)-catalyzed reaction can be the second step of sequence two: glycerol kinase
ATP + glycerol 98 glycerol 3-phosphate + ADP (5) The glycerol 3-phosphate is oxidized by the dissolved oxygen content of the medium in a reaction catalyzed by the enzyme glycerophosphate oxidase (EC 1.1.3.21). glycerophosphate oxidase
glycerol 3-phosphate + O2 98 dihydroxyacetone phosphate + H2O2 (6) Reaction sequence two was studied by Wimmer et al.,24 who worked out a sensitive colorimetric procedure to replace the (21) Moss, D. W.; Henderson, A. R. In Tietz Textbook of Clinical Chemistry, 2nd Ed.; Burtis, C. A., Ashwood, E. R., Eds.; W. B. Saunders & Co.: Philadelphia, PA, 1994; pp 797-825. (22) Tsuchida, T.; Yoda, K. Clin. Chem. 1983, 29, 51-55. (23) Nguyen, V. K.; Wolff, C. M.; Seris, J. L.; Schwing, J. P. Anal. Chem. 1991, 63, 611-614. (24) Wimmer, C. M.; Artiss, J. D.; Zak, B. Clin. Chem. 1985, 31, 1616-1620.
Figure 6. Creatine kinase response: current-time response curves recorded with reaction sequence one. Ten microliters of Reagent-I, 10 µL of creatine phosphate solution (CP-I), and 80 µL of creatine kinase enzyme solution (sample) or buffer were mixed. A 15-µL aliquot of this mixed reaction medium is added onto the disk. Sample activities: (a) 1500, (b) 1000, (c) 500, (d) 300, (e) 150, and (f) 100 munit/mL; (g) reagent blank.
Figure 7. Creatine kinase calibration curve for reaction sequence one: current-time response slopes vs creatine kinase enzyme activity calculated from the transient curves shown in Figure 6.
generally used UV spectrophotometry with simple colorimetric detection of total creatine kinase activity. A peroxidase-catalyzed final color-generating step was needed. In our experiments, both sequences were investigated. Measurements with Reaction Sequence One. To observe the CK response of the cell, two separate reagent solutions (Reagent-I and CP-I) were prepared. One milliliter of the Reagent-I solution contained 10.2 mg of AMP, 5 mg of ADP, 3.7 mg of creatinase, and 3.7 mg of sarcosine oxidase in pH 6.7 HEPES buffer. One milliliter of CP-I solution contained 62.5 mg of creatine phosphate in HEPES buffer. Adding the necessary three solution components of the reaction media (two reagents and the sample) into the cell, current-time curves with reaction-rate-dependent slopes could be recorded. Figures 6 and 7 show response slope-CK activity dependence. In these measurements, 15-µL total solution volumes were added onto the reaction disk punched out of Olympus lens cleaning cloth. The 15-µL solution aliquot was taken out from a premixed reaction medium containing 10 µL of Reagent-I, 10 µL of CP-I, and 80 µL of CK enzyme or buffer solution. The cell gives a well-defined response to CK enzyme activity. The difference between the slope of the reagent blank “response curve” and that of a solution of small enzyme activity, 100 munits/mL, is
Figure 8. Creatine kinase current-time response curves recorded with reaction sequence two: 4 µL of Reagent-II, 4 µL of creatine phosphate solution (CP-II), and 2 µL of enzyme solution of different enzyme activities were added on to the disk. CK activities: (a) 1.96, (b) 1.31, (c) 0.87, (d) 0.43, (e) 0.21, and (f) 0.11 unit/mL; (g) reagent blank.
still apparent. Repeating the measurement 11 times with 280 munits total CK enzyme activity added on to the reaction disk, and using 18 µL total solution volume, a 0.64 nA/s average slope of the response curves was obtained, with a relative standard deviation of 8.4%. The reproducibility of the 200-s current values between electrochemical cells was about the same (6-8%) as that obtained by repeating the measurements with the same cell. We assume that the reproducibility of the method is primarily determined by the reproducibility of the sample introduction and handling using micropipets. Measurement with Reaction Sequence Two. The composition of the reagent mixture was selected on the basis of the colorimetric study of Wimmer et al.24 Based on our previous studies, the creatine phosphate component was separated from the other reagent components and applied as a separate solution. A 1-mL aliquot of Reagent-II contained 5.6 mg of AMP, 3.84 mg of ADP, 1.5 mg of L-R-glycerophosphate oxidase, and 0.1 mg of glycerol kinase in pH 6.7 HEPES buffer. The concentration of the CP solution (CP-II) was 11.5 mg/mL. Reaction sequence two also gave good CK enzyme activitydependent signals using the amperometric microcell. In our studies, several membrane materials and various reagent and sample solution volumes were used to collect information about the influence of the different parameters on the signal. In the response curves shown in Figure 8, 4 µL of Reagent-II and 4 µL of CP-II substrate were added on the Olympus lens cleaning tissue reaction disk. After a steady and measurable background current (15-40 nA) was achieved, 2 µL of CK enzyme or buffer solution was spread on the disk. The slope values of the response curves are plotted against the enzyme activity in Figure 9. CONCLUSIONS Our preliminary results show that the described amperometric microcell can provide enzyme activity-dependent signals in sample volumes as small as a few microliters. They suggest that the flatform microcell construction with the versatile reaction film and with selective hydrogen peroxide detection can considerably Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
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biotechnology. Applicability of these principles to ELISA can also be easily foreseen. If impregnated filters, containing all reagents in lyophilized form for the chemical reactions, can be prepared, then the new amperometric microcell will gain further application as a “reagentless” biosensor. In our laboratory, further work is in progress to exploit these possibilities. A simple, sensitive enzyme activity measuring cell can gain broad application in enzyme activity measurements as well as in enzyme-labeled immunoanalysis.
Figure 9. Creatine kinase calibration curve for reaction sequence two: current-time response slopes vs creatine kinase enzyme activity obtained from the data shown in Figure 8.
improve the competitiveness of amperometry in enzyme activity determinations. It can be expected that, by optimizing the procedures, reagent compositions, and devices for convenient sample dispensation, useful and practical analytical devices with cost-efficient operation can be developed for clinical diagnosis or
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ACKNOWLEDGMENT This work was supported by NSF/Whitaker Foundation Grant BES-9520526. We thank Mr. Stefan Ufer of the Biomedical Microsensors Laboratory, North Carolina State University, for the microfabrication of the base electrodes, and we acknowledge partial support by the NSF Engineering Research Center Grant CDR-8622201.
Received for review November 5, 1997. February 17, 1998. AC971221Z
Accepted
