Development of Amperometric Biosensors for the

free to common electroactive species and were success- fully applied for the ..... found to play an important role to both the sensitivity and the wor...
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Anal. Chem. 2002, 74, 132-139

Development of Amperometric Biosensors for the Determination of Glycolic Acid in Real Samples Constantinos G. Tsiafoulis, Mamas I. Prodromidis, and Miltiades I. Karayannis*

Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, 45 110 Ioannina, Greece

The first enzyme-based biosensors capable of determining glycolic acid in various complex matrixes, such as cosmetics, instant coffee, and urine, are presented in this paper. Two separate designs, both based on a threemembrane configuration consisting of an inner cellulose acetate membrane (CA) and an outer polycarbonate membrane (PC), which sandwich a membrane bearing the biomolecule(s), are proposed. Glycolate oxidase was immobilized onto a modified polyethersulfonate membrane by means of chemical bonding, and glycolate oxidase/catalase enzyme mixture was immobilized into a mixed-ester cellulose acetate membrane through physical adsorption. The membrane assemblies were mounted on an amperometric flow cell (hydrogen peroxide detection at a platinum anode poised at +0.65 V vs Ag/AgCl/3 KCl) or on an oxygen electrode, respectively. Both configurations were optimized with respect to various working parameters. The proposed biosensors are interferencefree to common electroactive species and were successfully applied for the determination of glycolic acid in various samples, showing an excellent agreement with a reference photometric method. The validity of the proposed method in samples, in which the reference method was not applicable, was tested with recovery studies. Values of 102 ( % were obtained. Inherent interference of oxalic acid was manipulated by using a primary aminecontaining buffer and the enzyme catalase. Both systems were designed in order to be compatible with the current technology of the most widely used commercial analyzers. Glycolic acid is a constituent of sugar cane juice. It is widely used in industry, especially in the processing of textiles, leather, and metals, wherever a cheap organic acid is needed, for example, in the manufacture of adhesives, in copper brightening, decontamination cleaning, dyeing, electroplating, in pickling, cleaning, and chemical milling of metals.1 For general-purpose uses, glycolic acid is the objective of much current research in many scientific fields, such as environmental chemistry (where it is recorded as one of the species present at elevated concentration in aerosols after the passing of biomass burning plumes over the tested site),2 microbiology (in which the glycolate test is a method to discriminate N-acyl groups of muramyl residue in peptidoglycan of bacterial cell walls),3 tissue * Corresponding author. Fax: +30-651-98796. E-mail: [email protected]. (1) Budavari, S., Ed. The Merck Index., 12th ed.; Merck & Co., Inc: Rahway, NJ, 1996; p 766.

132 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002

engineering (for the production of biomaterials based on lactic and glycolic acids copolymers),4 and toxicology (in which glycolic acid is the major toxic metabolite of ethylene glycol in serum and of dichloroacetic acid in blood and urine). Therefore, a suitable analytical method is necessary for clinical use in instances of ethylene glycol5 and of dichloroacetic acid6 intoxication. Glycolic acid is also an important indicator in urology, because primary hyperoxaluria type I, an inborn error of glyoxylate metabolism, is characterized by excessive synthesis of oxalate and glycolate. Glycolic acid has also been identified as a major oxalate precursor,7 and its assay is important in the differentiation of type I and type II primary hyperoxaluria and in the evaluation of patients with the pyridoxine-responsive variant of primary hyperoxaluria type I.8 A possible relationship of protein intake to urinary glycolate excretion9 has also been reported and further strengthens the necessity of a fast, reliable, and easy-to-use analytical method for the assay of glycolic acid in urine. R-Hydroxy acids, and particularly glycolic acid, are used in many cosmetics products as exfoliants and moisturizers. Glycolic acid acts as a solvent for the intercorneocyte matrix, reducing excessive epidermal keratinization, and has a beneficial action for the renewal of epidermis and the reduction of wrinkles.10-12 Moreover, glycolic acid efficiently increases skin elasticity as a result of direct stimulation during the production of collagen, elastin and mucopolysaccharides.13 A comparative study regarding the effectiveness of a number of R-hydroxy acids, including glycolic, lactic, citric, hydroxybutyric, and malic acids with respect (2) Jaffrezo, J.-L.; Davidson, C. L.; Kuhns, H. H.; Bergin, M. H.; Hillamo, R.; Maenhaut, W.; Kahl, J. W.; Harris, J. M. J. Geophys. Res. 1998, 103, 3106731078. (3) Uchida, K.; Kudo, T.; Suzuki, K.-L.; Nakase, T. J. Gen. Appl. Microbiol. 1999, 45, 49-56. (4) Chevallay, B.; Herbage, D. Med. Biol. Eng. Comput. 2000, 38, 211-218 and references therein. (5) Porter, W. H.; Rutter, P. W.; Yao H. H. J. Anal. Toxicol. 1999, 23, 591597. (6) Narayanan, L.; Moghaddam, A. P.; Taylor, A. G.; Sudberry, G. L.; Fisher, J. W. J. Chromatogr. 1999, 729, 271-277. (7) Liao, L. L.; Richardson, K. E. Arch. Biochem. Biophys. 1972, 153, 438-448. (8) Williams, H. E.; Smith, L. H. The Metabolic Basis of Inherited Disease, 4th ed.; Stanbyry, J. B., Wyngaarden, J. B., Frederickson, D. S., Eds.; McGrawHill: New York, 1978; pp 182-204. (9) Holmes, R. P.; Goodman H. O.; Hart, L. J.; Assimos, D. G. Kidney Int. 1993, 44, 366-372. (10) Van Scott, E. J.; Yu, R. J. Arch. Dermatol. 1974, 110, 586-590. (11) Van Scott, E. J.; Yu, R. J. Can. J. Dermatol. 1989, 1, 108-112. (12) Cotellessa, C.; Peris, K.; Chimenti, S. J. Eur. Acad. Dermatol. Venereol. 1995, 5, 215-217. (13) Ditre, C. M.; Griffin, T. D.; Murphy, G. F.; Sueki, H.; Telegan, B.; Johnson, W. C.; Yu, R. J.; Van Scott, E. J. J. Am. Acad. Dermatol. 1996, 34, 187195. 10.1021/ac0106896 CCC: $22.00

© 2002 American Chemical Society Published on Web 12/01/2001

to their stinging potential on sensitive skin, their ability to increase skin cell renewal, and their ability to improve moisture content and to reduce lines and wrinkles characterizes glycolic and lactic acids as the most effective materials for developing R-hydroxy acid products.14 As a result of these properties, a wide variety of commercial cosmetics containing 8-35% w/w glycolic acid are available in different forms (cream, lotion, gel, and oil) and for different therapeutic targets (skin smoothing, face and body care, acne treatment, exfoliation process, photoaging care, skin renewal peel, moisturizing, sun protection, etc).15 Products used by physicians to perform chemical skin peeling contain up to 70% w/w glycolic acid. Unfortunately, the greater the glycolic acid cosmetic benefits, the greater is the potential for skin irritation, as reported by the U.S. Food and Drug Administration (U.S. FDA).16 Hence, the assay of glycolic acid in finished products is particularly important both for quantity control and for consumer safety. The determination of glycolic acid is also important in the food industry as part of the general concern for the determination of carboxylic acids.17 It is present in old wine, beer,18 fresh fruits, cane juice, instant coffee,19 molasses, and whey powder hydrolysates.20 The U.S. FDA also permits the use of glycolic acid for the cleaning of equipment used in the processing of meat, poultry, and egg products. The limited number of available methods for the determination of glycolic acid mainly includes gas chromatography/mass spectrometry,5 HPLC,6 ion-exchange HPLC,18,19 ion-exclusion chromatography,20 and reversed-phase ion-pair HPLC.21 These methods have inherent advantages (multianalyte analysis) and disadvantages (require complex isolation, derivatization steps, and expensive instrumentation); however, they do not provide the analytical simplifications of biosensors. Other photometric enzymic methods utilizing soluble glycolate oxidase are based either on the monitoring of the formed hydrogen peroxide, which is treated with the Trinder reagents,22,23 or on the monitoring of the formed glyoxylate using phenylhydrazine and ferricyanide.24 These methods are not suitable for routine analysis, because they are time-consuming, and they need sample pretreatment with active charcoal. Published work on the biosensor concept is also limited (from 1986 to date, only four papers have been published on this subject) and has mainly been focused on sensor demonstration rather than on the analytical applicability in real samples. Turner and co(14) Smith, W. P. Int. J. Cosmet. Sci. 1996, 18, 75-83. (15) (a) VanScott, E. G.; Yu, R. J. Alleviating Signs of Dermatological Aging with Glycolic Acid, Lactic Acid, or Citric Acid. U.S. Patent No. B1 5,547,988, August 20, 1996. (b) VanScott, E. G.; Yu, R. J. Method of Using Glycolic Acid for Treating Wrinkles. U.S. Patent No. B1 5,389,677, January 31, 1995. (c) U.S. Patent No. B1 5,385,938. (d) Web site of NeoStrata Company, Inc., http://www.neostrata.com. (16) Office of Cosmetics Facts Sheet; U.S. Food and Drug Administration; U.S. Government Printing Office: Washington, DC, 1995. (17) Blanco Gomis, D.; Mangas Alonso, J. J. Analysis for Organic Acids; Nollet, L. M. L., Ed.; Marcel Dekker Inc: New York, 1996; Vol. 1, pp 715-743. (18) Klein, H.; Leubolt, R. J. Chromatogr. 1993, 640, 259-270. (19) Badoud, R.; Pratz, G. J. Chromatogr. 1986, 360, 119-136. (20) Bipp, H. P.; Fischer, K.; Bieniek, D.; Kettrup, A. Fresenius’ J. Anal. Chem. 1997, 357, 321-325. (21) Scalia, S.; Callegari, R.; Villani, S. J. Chromatogr. 1998, 795, 219-225. (22) Kasidas, G. P.; Rose, G. A. Clin. Chim. Acta 1979, 96, 25-36. (23) Bais, R.; Nairn, J. M.; Potezny, N.; Rofe, A. M.; Conyers R. A. J.; Bar, A. Clin. Chem. 1985, 315, 710-713. (24) Maeda-Nakai, E.; Ichiyama, A. J. Biochem. 2000, 127, 279-287.

workers25 immobilized glycolate oxidase onto ferrocene-modified carbon paste electrodes, thus revealing the suitability of the specific mediator in a number of oxidases. Using the same mediation system, Oungpitat and Alexander26 developed a planttissue electrode for the assay of glycolate in urine samples that were treated with active charcoal prior to analysis in order to manipulate the interference of ascorbic acid. An amperometric glycolate sensor based on glycolate oxidase and electron-transfer mediators was developed by Hale et al.,27 but its analytical applicability was not tested. Recently, a chemiluminescene flow method for glycolate, based also on the concept of a plant-tissue biosensor, was proposed by Li et al.28 In this work, the selectivity is limited because of the multienzyme systems present in the tissue; interference problems of reducing compounds on the detection system have also not been addressed.28 Evaluating the already existing biosensors, it is obvious that there is still a need for improved glycolate biosensors, mainly in terms of analytical applicability. In this work, we propose the development of a glycolic acid enzyme electrode based either on a multimembrane configuration with immobilized glycolate oxidase in combination with a flow hydrogen peroxide detection system or on a multimembrane configuration with an enzyme system of coimmobilized glycolate oxidase and catalase in combination with a Clark-type oxygen electrode. Both systems are interference-free to common electroactive species, fast (no sample treatment is necessary), reliable, easy to use, and cost-effective. They have been tested on a variety of real samples, such as instant coffee, various forms of cosmetics, and urine for which, as mentioned above, glycolate assay is important. In our opinion, the present work offers a true alternative to the existing chromatographic methods, incorporating the simplicity and advantages of biosensors. EXPERIMENTAL SECTION Chemicals. Glycolate oxidase (GlOD, EC 1.1.3.15, from spinach, 10 U/mg, as suspension in a 3.2 M (NH4)2S04 solution containing 2 mM FMN), catalase (CAT, EC 1.11.1.6, from bovine liver, 46500 U/mg, as suspension in an aqueous 0.1% thymol solution), glycolic acid, glyoxylic acid, and cellulose acetate (∼40% acetyl), were obtained from Sigma (St. Louis, MO). Polyvinyl acetate (PVA, MW 167 000 Da) and 2,7-dihydroxynaphthalene were supplied from Aldrich (Gollingham, Germany) and Fluka (Buchs, Switzerland), respectively. All other (analytical grade) chemicals were purchased from Sigma. Diluted catalase solution was prepared by diluting 100 µL of the bulk solution with 5 mL of 50 mM phosphate buffer, pH 7 (immobilization buffer). Membranes. Immunodyne ABC (nylon 66; preactivated; 0.45 µm porosity; thickness, 120 µm) and UltraBind membranes (modified polyethersulfonate membrane possessing aldehyde functional groups; inert to moisture and heat, thus eliminating the need for special storage; preactivated; 0.45 µm porosity; thickness, 150 µm) were the kind gift of Pall Filtration (Milan, Italy). MF membranes (mixed ester cellulose; of 0.45 µm porosity; (25) Dicks, J. M.; Aston, W. J.; Davis, G.; Turner, A. P. F. Anal. Chim. Acta 1986, 182, 103-112. (26) Oungpipat, W.; Alexander, P. W. Anal. Chim. Acta 1994, 295, 37-46. (27) Hale, P. D.; Inagaki, T.; Sullee, H.; Karan, H. L.; Okamoto, Y,; Skotheim, T. A. Anal. Chim. Acta 1990, 228, 31-37. (28) Li, B.; Zhang, Z.; Jin, Y. Anal. Chem. 2001, 73, 1203-1206.

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Figure 1. (A) Schematic representation of FI manifold employed for glycolic acid determination. (B) Assembly of the biosensors: LM, large molecules; AN, analyte; IN, interfering agents; CA, cellulose acetate membrane; EM, enzyme membrane (Ultrabind/GlOD for flow method; MF/GlOD/CAT for oxygen electrode; PC, polycarbonate membrane).

thickness, 115 µm) were purchased from Millipore (Poole, U.K.). Polycarbonate membranes (thickness, 10 µm; 0.03, 0.05, and 0.1 µm porosity) were supplied by Nucleopore (Cambridge, MA). For casting cellulose acetate membrane, a wet film applicator (5 in.; 1-8 mils; URAI s.p.a.; Milan, Italy) was used. Apparatus. The work was carried out using an in-house fully automated FI manifold29 equipped with a resident program, synchronized with the acquisition software (GPES3, Eco Chemie, Utrecht, The Netherlands). Electrochemical experiments were run using a computer-controlled potentiostat (Eco Chemie/Autolab). Solutions were pumped through using a four-channel peristaltic pump (Gilson, France), and sample injections were made using a pneumatically actuated injection valve (Rheodyne, Cotati, CA). A three-electrode flow-through detector (Metrohm, Switzerland) was used for the amperometric monitoring of hydrogen peroxide. This comprises respectively of a wall-jet thermostated cell (volume < 1 µL) with a platinum (L, 1.6 mm; BAS, West Lafayette, IN) working electrode, a built-in gold auxiliary electrode, and a Ag/ AgCl/3 M KCl reference electrode. A flow diagram of the system is shown in Figure 1. An oxygen electrode assembly from Rank Brothers Ltd. (Bottisham, U.K.) comprising of a central platinum disk (L, 2 mm) and 1-mm-wide silver ring (pseudoreference electrode) was used for the monitoring of oxygen depletion.30 Preparation of the Enzymic Membranes. GlOD on Ultrabind or ImmunodyneABC membranes and the GlOD/CAT enzyme mixture in MF membranes were immobilized by covalent bonding and physical adsorption, respectively, following the immersing method.31 A 0.6-cm-diameter piece of the membrane was immersed in the immobilization solution for 3 h under mild stirring: flow method, 50 µL (1.4 U) of the GlOD solution in 0.5 mL of the immobilization buffer; oxygen electrode, 50 µL (1.4U) of the GlOD solution and 12 µL (214U) of the diluted CAT solution in 0.5 mL of the immobilization buffer. In both cases, unattached protein was removed by washing the membranes (3 × 10 min) with the immobilization buffer, and the membranes were then stored in the immobilization buffer at +4 °C. Assembly of the Sensor. The cellulose acetate membrane (20 µm thick, 100 Da nominal MW cutoff) was first placed on the platinum surface to eliminate interference from electroactive species. This membrane was prepared in our laboratory by (29) Prodromidis, M. I.; Tsibiris, A. B.; Karayannis, M. I. J. Autom. Chem. 1995, 17, 187-190. (30) Klemm, J.; Prodromidis, M. I.; Karayannis, M. I. Electroanalysis 2000, 12, 292-295. (31) General Protocols for Binding of Proteins on PALL Immunodyne Immunoaffinity Membranes; Pall BioSupport: Portsmouth, U.K.; 2001.

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dissolving 3.96 g of cellulose acetate and 4 mg of PVA in a mixture of 60 mL of acetone and 40 mL of cyclohexanone.32 Then the membrane bearing the enzyme(s) was superimposed with an outer polycarbonate membrane (0.05 µm in flow experiments, 0.1 µm in oxygen electrode) in order to prevent microbial attack and also leaching of the enzyme(s). All of the membranes were tightly fitted over the electrode with the aid of an O-ring (Figure 1).32 Sample Preparation. Coffee samples were prepared by diluting 2-4 g of the product in 100 mL of distilled water. Threefold diluted urine samples were used without any pretreatment (proposed method) and after treatment with active charcoal (Meck, Product no. 2183) for 15 min at 40 °C (validity experiments). Cosmetic samples in cream, lotion, and gel forms were prepared by transferring 0.5-1.0 g of the product into a 100 mL volumetric flask containing ∼60 mL of distilled water. The volumetric flask was then placed in a water bath at 60 °C for 15 min and swirled frequently. After cooling to room temperature, the flask was filled up to the line. Cream samples were centrifuged for 10 min at 5000 rpm. Peel samples were prepared by diluting 200 µL of the product in 100 mL of distilled water. Procedures. Flow Method. The carrier stream (50 mM phosphate, pH 7, containing 1 mM FAD) was continuously pumped at a flow rate of 0.42 mL min-1 toward the probe until a stable baseline current was reached (1-2 nA, within 20-30 min). Standard or sample solutions of glycolic acid [x µL sample + (2500 - x) µL carrier] were introduced as short pulses of 120 µL via the loop injection valve. The peak height of the current response was taken as a measure of the analyte concentration. Oxygen Electrode. A (2.5 - x)-mL portion of 150 mM Tris buffer, pH 8.3, was introduced into the reaction cell of the oxygen electrode assembly, and the pressure of the oxygen in the cell was allowed to reach equilibrium with the atmospheric oxygen under moderate stirring. When a stable current value was observed, appropriate values (x mL) of glycolic acid were added, and the current change after 1 min was taken as a measure of the analyte concentration. RESULTS AND DISCUSSION Enzyme Pathway. Glycolic oxidase is a peroxisomal enzyme that catalyzes in the presence of oxygen the conversion of glycolic acid to glyoxylic acid and hydrogen peroxide (Scheme 1). The isolated spinach enzyme was reported to have a subunit molecular weight of 43 000 Da and was enzymatically active only as tetramers (32) Prodromidis, M. I.; Tzouwara-Karayanni, S. M.; Karayannis, M. I.; Vadgama, P. M. Analyst (Cambridge, U.K.) 1997, 122, 1101-1106.

Scheme 1 . Enzymic Pathway of the Oxidation of Glycolic Acid

Table 1. Loading Test of GlOD onto Ultrabind Membrane units membrane

before

after

immobilized

rel efficiency, %

1 2 3 4 5 6

0.28 0.56 0.84 1.12 1.38 1.66

0 0 0.06 0.17 0.30 0.30

0.28 0.28 0.78 0.95 1.08 1.08

16 54 72 88 100 100

or octamers.33 The protein tended to irreversibly aggregate in an inactive form, especially in the absence of added FMN, one equivalent of which binds weakly and reversibly to each subunit. In subsequent undesirable reactions, enzyme-generated glyoxylic acid can react with the produced H2O2 to yield formic acid and CO2,34 or it can be further oxidized by glycolate oxidase and oxygen to produce oxalic acid35 under a slower reaction kinetic, as compared to that of glycolic acid. Enzyme Membranes. Previous studies of the tested membranes have shown that the spot-wetting immobilization procedure is very effective; however, under the specific conditions, it seems to be ineffective, because the high ionic strength of the enzyme solution, 3.2 M (NH4)2SO4, allows partial wetness of the membrane. A suggested procedure of membrane prewetting31 gave poor results. Dialysis of the enzyme solution was not tried, because the enzyme activity of GlOD has been found to be unstable in solution, especially in the absence of the appropriate preservatives.37 Enzyme loading for GlOD was tested in order to define the enzyme loading necessary to obtain diffusional limitation of response, that is, the response maximum. A saturation study of the membrane was made using the Ultrabind membrane, which claims to have the greatest binding capacity (135 µg IgG/cm2).31 Results of the enzyme-loading test are shown in Table 1. Experiments were then performed applying the useful enzyme content (33) Frigerlo, N. A.; Harbury, H. A. J. Biol. Chem. 1958, 231, 135-137. (34) Walton, N. J. Planta 1982, 155, 218-224. (35) Richardson, K. E.; Tolbert, N. E. J. Biol. Chem. 1961, 236, 1280-1284. (36) Price, N. C.; Stevens L. Fundamentals of Enzymology, 2nd ed; Oxford Science Publications: Oxford, 1989; p 143 (37) Zelitch, I.; Ochoa, S. J. Biol. Chem. 1953, 201, 707-718.

(1.4 U GlOD) onto other membranes. The relative apparent measurable efficiencies with respect to the most active membrane were 95, 42, and 100% for Ultrabind, Immunodyne ABC, and MF membranes, respectively. The results reveal that either the applied immobilization procedure is most suitable for the MF membrane or the chemical bonding in the ultrabind membrane affects, to a small degree, the conformation of the active site of the GlOD. Further experiments were carried out using an Ultrabind membrane for the flow detection of hydrogen peroxide (Ultrabind membrane has a porosity of up to 85% and a water flow rate of >56 mL/min cm2; porosity of MF membrane is 79% and provides a water flow rate of 38.5 mL/min cm2) and an MF membrane for the monitoring of oxygen depletion. The difference in response characteristics is in accordance with the kinetic parameters (Km) calculated by applying the electrochemical Eadie-Hofstee (E-H) and the direct linear (DL) transformations, using the equations

I ) Imax - Km(I/C) and

Imax ) I + (I/C)Km respectively. Data were treated according to the theory.36 The Km values found for ultrabind membrane are 1.09 (E-H) and 0.92 (DL) mM, and slightly lower values were found for the MF membrane, that is, 1.08 (E-H) and 0.85 (DL) mM. Results show an expected loss of the enzyme affinity, as compared with the literature value for the soluble enzyme (0.38 mM).38 Compared with previous studies, the values are slightly higher than those found for a plant-tissue based ferrocene-modified carbon paste electrode (0.62 mM).26 However, they are considerably lower than those reported for a carbon paste electrode containing siloxane polymer (2.2-4.8 mM)27 and those reported for a carbon paste electrode containing 1,1-dimethylferrocene as a mediator behind a dialysis membrane (13 mM).25 Optimization of Working Parameters in Flow Experiments. All of the experiments were carried out at an overall flow (38) Barman, T. F. Enzyme Handbook; Sringer-Verlag, Inc.: New York, 1969; Vol. 1, p 101.

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Table 2. Interference Effect of Various Compounds on the Assay of Glycolic Acid

Figure 2. Stability of immobilized GlOD at different membranes in the absence of FAD in the carrier stream: (9) Ultrabind, (b) Immunodyne ABC, and (2) MF. In the presence of 1 mM FAD in the carrier stream: (+) Ultrabind, (O) Immunodyne ABC, and (4) MF.

rate of 0.42 mL min-1, which reconciles fairly high peaks and satisfactory sample throughput (25 h-1) and a sample volume of 120 µL, because it prevents peak-broadening and ensures high sensitivity. Under the applied flow parameters, a dispersion coefficient of 1.21-1.25 was calculated. The pH of the working buffer was investigated in phosphate and Tris buffering systems covering the pH range 6-8. As expected, the latter gave lower responses, because it has been reported as a weak inhibitor of GlOD. The best results in terms of peak height were obtained at phosphate buffer pH 7 for the Ultrabind membrane and pH 7.5 for the MF membrane. The concentration of phosphate should be lower than 100 mM, because at higher concentrations, phosphates slightly inhibit the GlOD. Further experiments were performed at a 50 mM phosphate, pH 7, buffer solution. The effect of the cofactor FAD was also investigated and was found to play an important role to both the sensitivity and the working stability of the biosensor. Optimum working concentrations of FAD were determined for an Ultrabind membrane in a 50 mM phosphate buffer, pH 7 (as an added reagent in the carrier stream), and were found to be >1 mM (data not shown). This value refers to the concentration at which the corresponding curve reaches a plateau; subsequent work was carried out using 1 mM FAD. As reported in the literature, flavin mononucleotide (FMN) is the most effective prosthetic group for spinach glycolate oxidase. FAD is also active, although less so than FMN. The half-maximum reaction velocity required almost one-half the concentration of FMN, as compared with that of FAD.37 However, all of the optimization experiments were carried out with FAD, because its price is 200-fold lower than that of FMN, thus being conducive to the effective reduction of the overall cost of the method. As mentioned above, the presence of FAD in the carrier stream is beneficial for the working stability of the system. Figure 2 shows the dependence of the relative efficiency of the system on the number of injections. In the absence of FAD, the relative efficiency of the system decreases dramatically, retaining only 15% (MF and Immunodyne ABC membranes) of its original value after 60 injections. In the case of the Ultrabind membrane, the working stability is substantially improved, retaining almost 75% of its 136 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002

interfering species, mMa

rel efficiency, %

glycolic acid (0.25) acetic acid (10) adipic acid (10) Ascorbic acid (2) citric acid (10) formic acid (2) glutamic acid (5) glycerol (10) glyoxylic acid (0.5) lactic acid (0.5) lactic acid (1) malic acid (4) oxalic acid (2) succinic acid (10) tartaric acid (10) uric acid (2) Ca2+, Mg2+, K+ (2 each) Cl-, NO3-, NO2- (2 each)

100 95.4 95.5 101 104.6 110.7 100 98.2 83.1 105.2 133.8 107.3 42 98.2 98.3 101 100.3 100.5

a The values in parentheses are the millimolar concentrations of the compounds.

original value; however, recalibration between runs is necessary. No intermediate recalibration is necessary for all types of membranes when FAD is present in the carrier stream. Under these conditions, the system is quite stable (>90% relative efficiency) for more than 100-120 injections. Interference Study. The interference of some ions, electroactive compounds, and a big variety of organic acids present in real samples (with the exception of glyoxylate) was investigated by applying the method of mixed solutions in the presence of 0.25 mM glycolic acid. The effect of various compounds on the relative response is shown in Table 2 in which the activity of plain 0.25 mM glycolic acid was taken as 100%. The enzymic reaction suffers from interferences from glyoxylic acid and oxalic acid, because their presence in high concentration depresses the overall reaction to the left, according to the reaction Scheme 1. The interference of glyoxylic acid has no significance on the analytical applicability of the sensor, because it is not present in natural matrixes. On the other hand, oxalic acid interference is prohibitive for the application of the method in samples in which the concentration of oxalic acid is equal to or higher than that of glycolic acid, for example, urine. A slight positive interference from lactic acid is also expected when it is present in at least 2-fold concentration than that of the glycolic acid. This presumably can be attributed to its structural similarity to glycolic acid.37 A small increase of the system efficiency observed in the presence of formic acid can be attributed to limited production of carbon dioxide, which is an inhibitor of the GlOD activity.39 No interference was observed from common electroactive compounds, such as ascorbic and uric acids because of the existence of the cellulose acetate membrane. This is of great analytical interest for the applicability of the method in food and urine samples. Application to Cosmetics and Instant Coffee Samples. Under the optimum conditions, two calibration graphs, current/ nA ) f([glycolic acid/mM]), were constructed, applying the leastsquares method. Using the MF membrane, a calibration graph (39) Branden, R.; Styring, S. Biochem. Biophys. Res. Commun. 1979, 89, 607611.

Table 3. Flow Determination and Recovery Studies of Glycolic Acid in Cosmetics and Instant Coffee Samplesa recovery studies sample

proposed methodb (% w/w)

reference method (% w/w)

labelingd (% w/w)

rel error (%)

added (mM)

found (mM)

recovery (%)

smoothing creamc smoothing lotionc gel plusc skin renewal peelc coffee (Jacobs) coffee (Nescafe)

8.66 8.64 16.38 35.35 0.40 0.31

8.53 8.10 15.89 34.56

8 10 15 35

1.5 6.7 3.1 2.3

0.300 0.300 0.200 0.300 0.200 0.200

0.302 0.328 0.211 0.319 0.202 0.205

100.7 109.3 105.5 106.3 101 102.5

a The standard deviation of the mean ranges from 0.04 to 0.09 w/w. b Average of three runs. c Products of Neostrata Company Inc., Pricenton, NJ. d Glycolic acid content of the products according to the manufacturer.

Scheme 2. Reaction of a Primary Amine with Glyoxylic Acid

that was linear over the concentration range 0.01-0.6 mM glycolate was plotted. The equation for the straight line is y ) 0.34 + 61.44 [glycolate/mM], with a correlation coefficient r ) 0.998. The detection limit for a signal-to-noise ratio of 3 (S/N ) 3) was 5 µM glycolate. Using Ultrabind membrane, a linear relationship was obtained between the response and the glycolate concentration in the range 0.01-1 mM with a correlation coefficient, r ) 0.997. Data fit the equation y ) 0.52 + 51.34 [glycolate/mM]. The detection limit (S/N ) 3) was 6 µM glycolate, and the relative standard deviation (RSD) of the method was calculated as 1.8% (n ) 5, 0.25 mM glycolate). When not in use, enzyme membranes were stored at +4 °C in the immobilization buffer, and they retained almost 80% of their original activity after a period of three weeks. All assays were carried out using the Ultrabind membrane, and the results of the determination of glycolic acid in various formulations of cosmetics and instant coffee samples are summarized in Table 3. The results in cosmetic samples were compared with those obtained using the photometric method of Calkins.40 According to this method, glycolic acid is heated (3 h, 90 °C) with 2,7-dihydroxynaphthalene in concentrated sulfuric acid, developing a violet product (λmax, 545 nm), the absorbance of which is proportional to the concentration of glycolic acid. The mean relative error was 3.4%. Validation experiments were not performed for instant coffee samples because of the development of an intense brown color in the reaction tubes after the addition of sulfuric acid (partial decarbonization of the organic content), even in highly diluted samples. The accuracy of the method was, therefore, verified by recovery studies performed by adding standard glycolic acid solutions to samples. Recoveries of 100110% were achieved, as shown in Table 3. Optimization of the Working Parameters with the Oxygen Electrode. As mentioned above, the biosensor based on glycolate oxidase suffers from oxalate interference making it, thus, not applicable for use in urine samples, for which the determination of glycolic acid is very important for the diagnosis of primary hyperoxaluria. To overcome this problem, a new biosensor design is proposed in this work. This is based on the coimmobilization

of the enzymes glycolate oxidase and catalase operating in a primary amine-containing buffering system, and targeting the restriction of any undesirable side reactions, for example, the enzymatic oxidation of glyoxylate to oxalic acid and the chemical oxidation of glyoxylate to formic acid. The use of catalase aims at the decomposition of the enzyme-generated hydrogen peroxide in order to restrict further oxidation of glyoxylate to formate and to prevent glycolate oxidase from deactivation.41 This new system was investigated in the presence of certain primary amines, such as ethylenediamine (EDA) and Tris, in the presence of a dipeptide containing a terminal primary amine (glycylglycine), and in the presence of a tertiary amine (triethanolamine, TEA). According to previous studies by DiCosimo and co-workers,41 for the biocatalytic production of glyoxylic acid, primary amines are capable of reacting with glyoxylic acid to produce a mixture of the corresponding hemiaminal and imine, which limits further enzymatic oxidation of glyoxylic acid to oxalic acid or prevents its chemical oxidation to formate and carbonate (see Schemes 1 and 2). The same authors insist that the use of catalase or the use of a primary amine alone improves the yields of glyoxylate. Furthermore, the presence of these substances has an unexpected synergistic effect that favors the high production of glyoxylic acid, but the concentration of formic and oxalic acids is almost zero. In the present work, this new biosensor design was investigated in an oxygen electrode assembly, because the added catalase decomposes hydrogen peroxide, which was the measurable compound in the glycolate oxidase-based biosensor. The pH profile for a 50 mM phosphate buffering system and the effect of the FAD concentration were reexamined, and optimum values of 8 and 0.5 mM, respectively, were observed. The effect of the porosity of the outer polycarbonate membrane was also checked for values of 0.03, 0.05, 0.1, and 1.0 µm, considering as criteria the stability of the baseline, the sensitivity of the system, the (40) Calkin, V. P. Anal. Chem. 1943, 15, 762-763. (41) (a) Seip, J. E.; Fager, S. K.; Gavagan, J. E.; Anton, D. L.; Di Cosimo, R. Bioorg. Med. Chem. 1994, 2, 371-378. (b) Seip, J. E.; Fager, S. K.; Gavagan, J. E.; Gosser, L. W.; Anton, D. L.; Di Cosimo, R. J. Org. Chem. 1993, 58, 2253-2259.

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Table 4. Experimental Data Showing the Effect of Different Buffering Systems on the Response of GlOD and GlOD/ CAT Biosensors and the Oxalic Acid Interferences Using the Oxygen Electrode Assembly buffering systema

enzyme membrane

response to 0.25 mM glycolic acid (nA)

response to 0.25 mM glycolic acid and 1 mM oxalic acid (nA)

relative response (%)

50 mM phosphate, pH 7.5 50 mM phosphate, pH 7.5 50 mM TEA, pH 8.2 50 mM EDA, pH 8.3 50 mM Gly-Gly, pH 8.3 50 mM Tris, pH 8.3 150 mM Tris, pH 8.3 150 mM Tris, pH 8.3b 150 mM Tris, pH 8.3 150 mM Tris, pH 7.5 150 mM Tris, pH 7.5

GlOD GlOD/CAT GlOD/CAT GlOD/CAT GlOD/CAT GlOD/CAT GlOD/CAT GlOD/CAT GlOD GlOD/CAT GlOD

225 189 201 217 188 197 197 197 209 168 215

151 132 120 178 158 181 189 191 198 118 159

67 70 57 82 84 92 96 97 95 75 74

a

pKa(Tris) ) 8.1; pKa(TEA) ) 7.8; pKa(EDA) ) 6.85, 9.93; pKa(Gly-gly) ) 8.21. b In the presence of 2 mM formic acid.

retention of the immobilized enzymes, and the response time of the device. A polycarbonate membrane (10-µm thickness) of 0.1µm porosity was finally selected. Under these conditions, the behavior of the system was examined in the presence of catalase (MF-GlOD/CAT membrane); in the presence of an amine-containing buffer (MF-GlOD membrane); and in the presence of both (catalase and an amine containing buffer), taking the magnitude of the oxalate interference and the response of the system as a criterion. Table 4 summarizes the results obtained. A control reaction was performed using phosphate buffer, which lacks an amine functional group and so cannot react with glyoxylate to form hemiaminal or imine. The same behavior was also observed in the case of the tertiary amine, TEA. EDA, glycylglycine, and Tris (in the unprotonated form) gave better results, with the latter almost eliminating the interference effect of oxalic acid. Evaluating the results of Table 4, it is obvious that the oxalate interference can be effectively controlled in the presence of the unprotonated primary amine group and not in the presence of catalase. This statement is also supported by the decrease of the interference (4%) when the concentration of the Tris buffer, pH 8.3, is increased from 50 to 150 mM. However, the presence of catalase results in a much better background signal (smooth baseline increasing thus the S/N ratio) and slightly better results regarding the interference effect of oxalic acid (1-3%). In the presence of catalase, a 20% decrease in the response of the sensor was observed (see Table 4). This behavior was expected, because catalase competes for some of the immobilization sites in the MF membranes. The results are consistent with the expected behavior of an unprotonated amine, which forms an oxidation-resistant N-substituted hemiaminal or imine complex with glyoxylate. An amine buffer whose pKa is much higher than the pH of the reaction mixture would be present predominantly as the protonated amine in the reaction mixture, and therefore, it would be less likely to form such complexes with glyoxylate.42 The effectiveness of the new system in eliminating any side reaction during the catalytic oxidation of glycolic acid is further supported by the no-interference effect of formic acid when it is added at a concentration 8 times that of glycolic acid (see Tables 2 and 4). (42) Hoefnagel, A. J.; Van Bekkum, H.; Peters, J. A. J. Org. Chem. 1992, 57, 3916-3921.

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Table 5. Determination of Glycolic Acid in Urine Samples and Recovery Studies Using the Oxygen Electrodea

sample untreated treated no.1 treated no.2 b

recovery studies proposed reference rel methodb method error added found recovery (mM) (mM) (%) (mM) (mM) (%) 0.900 0.950 0.680

0.890 0.650

6.7 4.6

0.080 0.060 0.160

0.081 0.058 0.151

101 97.5 94.5

a The standard deviation of the mean ranges from 0.01 to 0.04 mM. Average of three runs.

Application to Urine Samples. Under the optimum conditions, a linear calibration graph, current/nA ) f([glycolic acid/ mM]), over the concentration range 0.02-0.8 mM glycolate was constructed, applying the least-squares method. Data fit the equation y ) 4.2 + 1290 [glycolate/mM] with correlation coefficient r ) 0.995. The detection limit (S/N ) 3) was 4 µM glycolate. The relative standard deviation (RSD) of the method was calculated as 1.6% (n ) 5, 0.25 mM glycolate). The results of the determination of glycolic acid in urine samples are summarized in Table 5. The results taken with urine samples (only these treated with active charcoal) were compared with those obtained with the photometric method of Calkins.40 (The detection limit (S/N ) 3) was 3 µM glycolate, and the RSD of the method was calculated as 1.8% (n ) 5, 0.025 mM glycolate). The accuracy of the method was also verified by recovery studies performed by adding standard glycolic acid solutions to the samples. Recoveries of 94-102% were achieved, as shown in Table 5. Membranes retain almost 90% of their original activity after 80-90 analytical runs. When not in use, they were stored at +4 °C in the immobilization buffer and retained almost 70-75% of their initial activity after a period of three weeks. CONCLUSIONS Both systems were developed in a manner to establish an easyto-prepare and -perform, cost-effective method for the determination of glycolic acid in real samples. The adapted technology of multi-membrane biosensors seems to be the most suitable for

reliable and interference-free amperometric measurements, and for this reason, it has found wide use in many biosensor based analyzers, for example, YSI 2700 Select Food Analyzer (YSI Inc., OH), ABD 3000 Biosensor Assay System (Universal Sensors Inc., Metairie, LA), BioProfile Chemistry Analyzer (Nova Biomedical, Waltham, MA), Alcohol Sensor (SensAlyse Ltd., Manchester, U.K.) etc. Both systems were found to be promising, and future work will be focused on their applicability to other real samples as well as to the optimization of storage conditions, that is, use of preservatives in order to increase the storage stability of the enzyme membranes. We believe that the proposed method could be the basis for improved biosensor-based analytical devices for

the assay of glycolic acid in real samples for which its determination is of great analytical importance. ACKNOWLEDGMENT The authors thank the Greek representative (Neogen, Ltd) of the Neostata Company Inc. for supplying us with the cosmetics samples. Thanks are also extended to L. Arbizzani, from Pall Italia srl., who kindly donated samples of the membranes. This work was supported by the Greek Ministry of Development (General Secretariat of Research and Technology), project PENED’99. Received for review June 19, 2001. Accepted October 1, 2001. AC0106896

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