www.elsevier.

nl/locate/jelechem Journal of Electroanalytical Chemistry 491 (2000) 182– 187

An electrochemical biosensor for formaldehyde
Y. Herschkovitz, I. Eshkenazi, C.E. Campbell, J. Rishpon *
Department of Molecular Microbiology and Biotechnology, Tel -A6i6 Uni6ersity, 69978 Ramat -A6i6, Israel Received 21 March 2000; received in revised form 15 April 2000; accepted 3 May 2000 Dedicated to Professor E. Gileadi on the occasion of his retirement from the University of Tel Aviv and in recognition of his contribution to electrochemistry

Abstract This paper reports the development of a novel detection method, based on the coupling of a biosensor measuring device and a flow-injection system, using the enzyme formaldehyde dehydrogenase and a Os(bpy)2-poly(vinylpyridine) (POs-EA) chemically modified screen-printed electrode. The sensor can detect 30 ng ml − 1 of formaldehyde in aqueous solution (corresponding to sub-ppb atmospheric concentrations of formaldehyde). The sensor is selective, inexpensive, stable over several days, and disposable, as well as simple to manufacture and operate. The system described here can easily be adapted to other substrates using their corresponding dehydrogenases. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Formaldehyde; Biosensors; Flow-cell; Formaldehyde dehydrogenase; Os(bpy)2-poly(vinylpyridine)

1. Introduction Since the industrial revolution, an unprecedented amount of hazardous air pollution (HAP) has been created. Currently, the largest source of HAP is automotive engine exhaust, which contains several toxic pollutants, such as formaldehyde. Formaldehyde, a widely used industrial chemical in many manufacturing processes, is toxic, allergenic and accumulates in the air over cities. Formaldehyde has been classified as a human carcinogen by both the US Environmental Protection Agency (EPA) and the World Health Organization [1,2]. Formaldehyde is released as a by-product of incomplete hydrocarbon combustion and is emitted at a rate of 700 mg l − 1 gasoline [3]. Approximately 4 × 1011 kg formaldehyde is formed in the troposphere annually through the photochemical oxidation of released hydrocarbons [2]. Most commercial formaldehyde is produced from methanol for use in many industrial processes, including wood fixatives; dry cleaning solutions; solvent use; boiler use; chemical production; oil, gas, and petroleum production, as well as paper and pulp production; the cosmetics industry and the textile
* Corresponding author. Tel.: + 972-3-6409836; fax: + 972-36409407.

industry [1,2,4,5]. In hospitals, formaldehyde is used as a disinfectant, as well as a fixative by pathologists, medical technicians, and researchers. Today, approximately 10 megatons per year of formaldehyde are produced [6]. Formaldehyde accumulates in the atmosphere over cities and is known to induce asthma-like symptoms in humans [7]. Polluted urban air contains between 0.010 and 0.160 ppm formaldehyde; in urban air on a sunny day, formaldehyde has a half-life of 50 min [8]. Exposure to formaldehyde can cause central nervous system damage; blood, immune system and developmental disorders; as well as blindness and respiratory disease [3,9,10]. Vapor-phase formaldehyde is linked to nasophrayngaeal carcinomas in rats. Standards have been set by the Occupational Safety and Health Administration, as well as by the EPA, to limit human exposure and health risk in occupations involving formaldehyde use. Formaldehyde levels must be accurately monitored to comply with these standards. Colorimetric detection methods, such as Deniges’ method, and Eegriwe’s, have been known since the beginning of the 20th century [5]. Unfortunately, these methods, reagents and reaction products are often just as harmful to human health and the environment as is formaldehyde. Currently, standard

0022-0728/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 0 0 ) 0 0 1 7 0 - 4

2. devised for this class of enzymes.1] (ADH) from bakers yeast (specific activity 450 U mg − 1). a flow-through detector monitors the products of the reaction. these methods are impractical for real-time measurements because of the required time for apparatus set-up. Both sample and reagent consumption are low and accuracy and precision are good. has recently been introduced [9. 2. The membrane was dried in air and then placed for 20 min in a blocking solution containing 0. the membrane was washed in 0.14] (SDH) from sheep liver (specific activity 6.1.1 M potassium phosphate buffer (pH 8) and kept at 4°C.46] from P. Recent efforts have turned toward the development of biological methods of detection combined with physical transducers. Some 250 NADH-dependent dehydrogenases and over 150 NADPH-dependent enzymes have been identified [16].16 – 21]. disposable sensor for NADH determination [22.24]. using analytical grade K2HPO4 and KH2PO4 purchased from Merck. either dry or in the buffer solution.13.1 M potassium phosphate buffer (pH 8). a carbon auxiliary electrode. 2.3. so that high sample throughput is possible [27]. The reaction products are measured before ‘steady-state’ conditions are established. FDH [EC 1. sorbitol dehydrogenase [EC 1. low-cost. Chemical modification of electrode surfaces bestows electrocatalytic properties to the electrode toward NADH electrochemical oxidation [12. a small.1.Y. and the readout is available within seconds of introducing the sample. the pollutant needs to be transferred from air to an aqueous solution and to be combined with an air sampling device [24. Electrochemical-based biosensors enable direct.1. putida (specific activity between 3 and 5 U mg − 1). Materials NAD+. Buffer solutions were prepared immediately before use. UK. as in standard air measurements. Chemically modified screen-printed electrodes (SPEs) enable the development of a reliable. NADH.1.1 M potassium phosphate buffer pH 8).32].2 U mg − 1). based on a biosensor using FDH and a chemically modified electrode. resulting in false positives. until use. abbreviated POs-EA. would be widely applicable and could provide a basis for enzyme-based electrodes for a variety of analytes. A general approach. Materials and methods 2. Immunodyne ® membrane preparation The FDH enzyme was immobilized onto the Pall Immunodyne® membrane (previously cut into 1. Dehydrogenase-based sensors have attracted attention because of the ubiquity of these enzymes. All reagents and electrolyte solutions were prepared using twice distilled water. formaldehyde (4% w/v). biosensors.2 × 1. alcohol dehydrogenase [EC 1.17]. Flow-injection systems have proved useful in practical applications in fields as diverse as water and environmental control and agricultural and pharmaceutical analysis [27 –31]. Immunodyne® ABC 5 mm cutoff. Polymers containing osmium compounds have been used as mediators for NADH electrooxidation in batch reactions. All other reagents and buffers were of analytical grade. Herschko6itz et al. Nylon membranes.23]. The redox polymerpoly(vinylpyridine) containing complexed (bpy)2OsCl groups and partially quaternized with bromoethylamine. gas chromatography and fluorimetry [11 – 15]. using immobilized glucose dehydrogenase for the determination of glucose [25. including formaldehyde using formaldehyde dehydrogenase (FDH).2. Finally. For air measurements.1. reliable. / Journal of Electroanalytical Chemistry 491 (2000) 182–187 183 formaldehyde-assessment methods include visible absorption.2. The electrodes were composed of a carbon working electrode. The sensor is designed to measure aqueous solution.1 M glycine in 0. were purchased from the Pall Corporation. precisely metered volume is ‘injected’ into a flowing stream containing the reagent. USA. in aqueous solutions based on the coupling of a biosensor measuring device and a flow-injection system. HPLC. Additionally. In a flow-injection system.26]. A limited number of devices for the real-time determination of formaldehyde in the gas phase. Screen -printed electrodes SPEs were purchased from Gwent Electronic Materials. In this article we present a novel approach for formaldehyde determination. sorbitol and glycine were purchased from Sigma.1. Further downstream. A successful combination between the reactions catalyzed by such enzymes and a transducer might therefore be expected to be of great importance and utility. and reproducible measurements [16.2 cm squares) by dropping aliquots (5 or 10 ml) of enzyme solution (30 mg ml − 1 in 0. was synthesized according to Gregg and Heller [33]. and an Ag . using immobilized FDH and a Os(bpy)2-poly(vinylpyridine) (POs-EA) modified SPE. All these methods require similarly toxic reagents and suffer from a number of interferences. and used according to the manufacturer’s instructions.

AgCl .

POs-EA solution (25 ml. The working electrode was modified using the redox polymer POs-EA crosslinked to a commercially available diepoxide poly(ethylene glycol) diglycidyl ether.1 M KCl reference electrode. 0. All working solutions contained 0. according to Gregg and Heller [33].1 M KCl. 4 mg ml − 1) was mixed with 5 ml of the diepoxide cross-linking .

1 M potassium phosphate buffer solution (pH 8). 2. The electrodes were connected to a computer-controlled BAS 100B potentiostat. Amperometric response of the SPE to successive additions of NADH. The working electrode was held at 350 mV versus Ag .184 Y. NADH + Osox “ NAD+ + Osred + H+ Osred “ Osox + 2e − (2) (3) Fig. The electrochemical flow cell The electrochemical system was based on amperometric measurements. using a 5 ml syringe. The potentiostat. (I) Screen printed electrodes. The electrochemical cell setup. The flow rate was optimized for each enzyme used. sorbitol. The electrochemical cell was washed with the working solution. The sensitivity of electrochemical detection depends upon the efficiency of the electron transfer from the NADH via the POs-EA mediator to the carbon electrode. sorbitol and alcohol determination Known concentrations of formaldehyde. 0. 1. Formaldehyde.1 M potassium phosphate buffer (pH 8).5. USA).1 M potassium phosphate buffer pH 8. A schematic diagram is shown in Fig. and then an aliquot of 5 ml was applied to the working electrode surface.5 mM NAD+ + 0. Results and discussion The formaldehyde biosensor was based on the following sequence of reactions [9]: HCHO + NAD+ + H2O “ HCOOH + NADH + H+ (1) Fig. connected to a syringe pump and to an injector equipped with a 5 ml injection loop.) (20 9 2°C) with a cover to protect from dust and light. 1. (II) The electrochemical flow cell. (20 9 2°C). 2. 3. the injector.t.4. The enzymatic membrane was placed directly onto the SPE.35 V. Eapp = 0. and the software were purchased from BAS Bioanalytical Systems (BAS. Numbers depict NADH concentrations. or ethanol in a solution containing 0. All measurements were performed at r. Fig. The electrode was left to dry overnight (16 h) at room temperature (r. Hence.t. we first established the optimal conditions of this reaction. / Journal of Electroanalytical Chemistry 491 (2000) 182–187 agent polyethylene glycol (PEG) (3 mg ml − 1). 2 depicts the amperometric biosensor response to successive injections of different concentrations of NADH (5 ml aliquots) into the flow system. 2.5 mM NAD+ + 0.1 M KCl + 0. Herschko6itz et al. 50 ml min − 1 flow rate. (– – –) unmodified electrode. The SPE was placed in a home- made micro (30 ml cell volume) flow cell. 0.1 M KCl. (— ) POs-EA modified electrode. The mixture was thoroughly mixed. 0. (III) The whole measuring system. were injected into the cell through the 5 ml injection loop.1 M KCl + 0.

The addition of formaldehyde resulted in a higher anodic current. due to the oxidation of NADH. The rapid response and the high sensitivity are clearly demonstrated. ranging from 30 ng ml − 1 to 4. in the presence or absence of 0.5 mg ml − 1. AgCl.5 mM formaldehyde. obtained in the flow cell with FDH immobilized on an Immunodyne® membrane. Although tails are observed on the de- . Fig. as shown in Eq. 4 shows the response of the biosensor to successive injections of different concentrations of formaldehyde. (3). Fig. 3 shows cyclic voltammograms of the POs-EAmodified electrode. Each peak represents the increase in current.

This value agrees with the formaldehyde data in the literature: 90 and 56 mM [9.1 M potassium phosphate buffer pH 8.21. did not significantly change the response. . Effect of NAD+ concentration on the sensor response. thus requiring further investigation. the current response decreased. At low concentrations of formaldehyde (30 ng ml − 1 to 1.994 and the Km calculated from the plot was 1. A Lineweaver –Burk plot resulted in a straight line with R 2 = 0. NAD+ is a known inhibitor of the direct electrochemical oxidation process of NADH [21].5 mM formaldehyde. the reaction between the mediator and NADH varied with the pH of the contacting buffer in an inverse manner. Detection of formaldehyde. 5 shows the dependence of the current response on the cofactor concentration. The stability of the immobilized enzyme on the Immunodyne® membrane is shown in Fig.1 M potassium phosphate buffer pH 8. inexpensive and portable flow cell. scending portion of the signal current.0 (data not shown). the current response showed a linear relation (y = 15. As has been observed in many studies. pH. concentrations are similar to literature values [9. pH 8. horizontal lines columns: 0.8 + 0. (– – –) 0. Eapp = 0. We further optimized the formaldehyde sensor performance by optimizing the cofactor concentration.24].5 mg ml − 1) injected into the electrochemical cell. / Journal of Electroanalytical Chemistry 491 (2000) 182–187 185 Fig.Y. and the optimal flow rate was 50 ml min − 1. At higher concentrations of NAD+ Fig. homemade.6 mg ml − 1 formaldehyde. so that the higher the pH. Fig.5 mg ml − 1 formaldehyde. The optimal NAD+ concentration found was 0. Cyclic voltammograms of POs-EA/PEG. the response was stable and linear over 7 Fig. 0. Dotted columns: 0.34. The optimal enzyme loading was 300 mg. no response to formaldehyde injection was observed. The optimal pH found by us was pH 8. Reducing the enzyme concentration on the membrane by half. This flow rate allowed enough time for the enzyme to react with both the substrate and the cofactor.35 V. which may also be true for the mediated process. (—) Background.5 mM. Enzyme loading 300 mg. 0. In control experiments without FDH.24]. As expected for an enzymatic reaction the linear relation does not hold at high formaldehyde concentrations.35]. 4.35 V.74 mg ml − 1 (58 mM).06x.997). R 2 = 0. flow rate and enzyme concentration. the lower the reaction rate. the response to formaldehyde is reproducible and linear. These tails could be due to air bubbles in the probably not ideal. from 300 to 150 mg. Numbers depict formaldehyde concentrations. Amperometric response of the sensor to injections of formaldehyde. Herschko6itz et al. When the membranes were stored at 4°C in potassium phosphate buffer.3 mg ml − 1 formaldehyde. modified SPE. 5. This phenomenon could be due to a pH-related change in the osmium mediator orientation on the electrode or to an inherent reaction mechanism [12. 3. 300 mg FDH immobilized on Immunodyne® membrane. 6. in the presence and absence of formaldehyde. Such. Eapp = 0. diagonal lines columns: 1.

wet membrane. We therefore examined the response of the sensor to methanol and found that the sensor retained its specificity for formaldehyde and did not respond to equivalent additions of methanol (data not shown). Fig. can be used for developing an ethanol biosensor [18.35]. 0. (B) Amperometric response to successive sorbitol injections. Coupling the electrocatalytic oxidation of NADH by the mediator to the reaction catalyzed by the NAD+dependent dehydrogenase enzymes enables the construction of amperometric biosensors for a large variety of other substrates [12.186 Y. resulted in a complete loss of bound enzyme activity and/or fouling of the membrane (data not shown).5 mM NAD+ + 0.t. Numbers depict substrate concentration in mg ml − 1. Herschko6itz et al. R 2 = 0.16.22. dry membrane. Dry storage of the immobilized membrane at r. / Journal of Electroanalytical Chemistry 491 (2000) 182–187 Fig. . 0.8 mg ml − 1. however. The data demonstrate the possibility of using such biosensors for the measurement of sorbitol.35 V. shown in Fig. Dried membranes stored sealed with silica gel at 4°C showed a linear response over a period of 7 days. . The simplicity of this system enables the quick preparation of an immobilized membrane. enzyme loading 300 mg.21].5x. Immobilized ADH. R 2 = 0.46 to 4. (A) Amperometric response to successive ethanol injections. day 7. 2. In fact.3 mg ml − 1 (y = 0. day 1. After 30 days. 7. Dry membrane. an important chemical in the food industry. day 1. Eapp = 0. a linear correlation between concentration and response Fig. . at increasing concentrations ranging from 46 ng ml − 1 to 7. 0. which is pertinent to fermentation.1 M pH 8. wet membrane. The successive addition of ethanol. however.992).9x. 7(B) depicts the response of SDH. to successive sorbitol additions.996). . Long-term stability of FDH immobilized on Immunodyne® membrane. . day 7. 6. days. day 30. commercial formalin contains methanol. only 6% of the initial enzymatic activity remained. 30% of the activity was lost. was achieved. Eapp = 0. The same design can be used with other dehydrogenase enzymes.5 mM NAD+. wet membrane. Detection of sorbitol and ethanol.6 + 7. This biosensor will be able to detect minute (10 − 9 g) amounts of alcohol using a small enzyme loading of 25 mg.74 + 2.35 V. at concentrations ranging from 0. The correlation between the increasing concentration of sorbitol and the current response resulted in a linear calibration curve (y = 7. At concentrations ranging from 46 ng ml − 1 to 2.1 M potassium phosphate buffer pH 8. which can retain its bound enzyme activity for over a week and thus is ideally suited for on-site measurements. 7(A). resulted in a parallel successive increase in current response. Methanol is often used as a stabilizer for formaldehyde solutions and can also be found in mixed-waste vapors containing formaldehyde [36].6 mg ml − 1. immobilized on the Immunodyne® membrane at 300 mg enzyme loading.

B. B. Du Pont de Nemours. S.C.L. and low cost. Chem. Chemical Research Division. Soc.P. 77 (1981) 314. 1953. L. Q. In our experiments. At higher concentrations. Shiels. Di Benedetto. Miller.J. D. Kiba. Suzuki. So. Faraday Trans.T.A. Boguslavsky. The detection limit of the sensor was 30 ng ml − 1 in the solution corresponding to 0.. Dasgupta. Haemmerli. Chem. P. Margesin. M. L. B. This detection limit is the lowest ever reported for formaldehyde detection using electrochemical and enzymatic methods [9. Chem. J. H. Hammerle.F. Y. J. Ellis. J. Chem.S. Mazur. Electroanal. [3] J. Sens. Curr. G. [2] World Health Organization.J. Dimitrakopoulos. Chim. To conclude. J. Narvaez. Conclusions In this work.A. A. P. 20 (1990) 54. Heller. Johansson. Chem.E.J. Electroanal. L.D. Hall.A. Anal.A. S. Gay. 364 (1994) 277. the formaldehyde-selective sensor did not respond to injections of methanol. Lewis. Anal. Technol. Environ. Sci. Formaldehyde.L. Chem. Okamoto. A. I. M. B.L. Z. the response was not linear but the sensor remained sensitive to increasing amounts of the substrate. M. Incorporating the sensor into the micro-flowinjection system enabled us to use very small amounts (sub-nanogram) of sample. WHO. P. Gorton. P.A. S. Manz. Durst. Talanta 43 (1996) 915. Koivusalo. Phys. H. Kitchens. Sequeira. The enzyme-modified membranes could be stored for 7 days at 4°C without losing activity. R. Miller. A. Bufalini. S. J. Garn. Sun. M. Clin.S. Edwards. and a micro flow-injection system allowed us to detect formaldehyde with the high sensitivity and selectivity that is required by the EPA and OSHA. Rigo. R. Chemilumin. Hall. M. Methods Enzymol. I. Riley. Skotheim. Sprules. M. Soc. B. Anal. Rocks. York. G. R. A. 66 (1994) 551. Kitani. Biosensors 3 (1988) 359. 56. an immobilized enzyme on an Immunodyne® membrane. Ludi.H.W. Am. Uotila. P. L. Sprules. M. D. Gorton. Yokose. together with reusability. N. Appl. A. Y. D. M.J. R. Chem. .D. L. L. Acta 378 (1999) 169. A novel combination of a POs-EA modified SPE. Hodgins. Acta 304 (1995) 17. L. Biotechnol. J. Actuators B 37 (1996) 49. 8 (1993) 183. E. Tunon. H. Dominguez. alcohol and sorbitol. Wring. Speiser. Price. selectivity.G. G. Anal. Fainman. Whitaker. M. emphasizing the formaldehyde detection. Lobo. Ha ¨ mmerle. Analyst 121 (1996) 1891. Pittson. second ed. 6 (1972) 816. Zawodzinski.L. Sens. three dehydrogenases were used. M. Persson. Anal.J.T. Opin. Environ. Despite the presence of methanol in commercial formalin solutions. J. Vering. 95 (1991) 5970. 53 (1981) 1979. H. NY. 5.W. 2 (1991) 9.J.J. 16 (1982) 543A.P. Hedenmo. Macri. S. Alexander.H. 25 (1992) 75.A. F. Scarpa. Investigation of selected potential environmental contaminants: formaldehyde. Fultz. T. DC. Worsfold. sensitivity.. Liolios. Health 54 (1999) 115. Persson. Hileman. J. J. 103 (1981) 7636. 1989. Herschko6itz et al. B. 1989. J.C. Ngila. A. J. Electrochem. 1992. Martos. M. Environ. E. J. Electroanal.D. S. Technol. Y. C.I.H.W. A. Family Health and Medical Guide.G. T. N. T.D. Formaldehyde.B.L. Brubaker. H. J. Widmer. Hibbert. H. Arch. Di Paolo. Biotechnol. Pletcher. Casner.N.B. American Chemical Society. W. E. W. Biosensors and Chemical Sensors.W. Elec- [29] [30] [31] [32] [33] [34] [35] [36] [37] trochemical Department. Anal. L. Kim. Biosensors 4 (1989) 61.5 mg ml − 1). 103 (1981) 3595. Jaegfeldt. A.A.J. Biosens.Y. L. P. Hart. US Environmental Protection Agency. T. Washington. Seiwald.I. 287 (1990) 61. Chim.L. [5] J. 292 (1990) 115. Haward. K. H. A. Electroanalysis 9 (1997) 191. Lan. L. .I. [4] L.24. 28 (1982) 409. Wring. Niagara Falls. Gorton. Hart. So.F. B. The sensor response was linear over a wide range of concentrations (30 ng ml − 1 to 1. Hale. 11 (1996) 239. Cho.L.A. Chasnoff. J.2 vppb concentration of the formaldehyde in the space above the formaldehyde solution [9].A. Y. Kazue. Hall. Soc.T.36. I. Ye. Analyst 119 (1994) 253. B. Bioelectron. Zen. E. Ch. formaldehyde dehydrogenase. B. Biolumin. Gorton. Lui. Am. Maple Press PA. Torstensson. P.D. J.S. Such small amounts are notably important when dealing with toxic chemicals. IL. R.M. Chem. D.J. 71 (1999) 1513.37]. Bartlett. we have presented a generally applicable approach and three examples that are based on the mediated oxidation of enzymatically produced NADH. Publications International. Actuators B 34 (1996) 516. S. S. / Journal of Electroanalytical Chemistry 491 (2000) 182–187 187 4. Fan. Lee. Kitani. Persson.K. Chem. H. J. Cade. J. Schumann. Chem. [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] References [1] National Air Quality and Emissions Trend Report: Air Toxics. M. Chim. US Environmental Protection Agency. Kim. M. Karan. Geneva. 1 (1986) 1245. B.H. Gregg. Walker. D.H. Miranda.I. Rishpon. 1976.19.A. Lincoln-Wood. Vianello. 204 (ARC-49-5681). Sci. Montenegro. Environmental health criteria 89. E. D. C. 1996. ADH and SDH for the respective detection of formaldehyde. J. Stefani. Acta 140 (1982) 1. Pawliszyn.O. Ch. Katakis. Soncini. the sensor described in this work assembles the basic requirements for an on-the-spot biosensor: simplicity. D. Rosen.E. Schmidt. 6. E. B. Gooding. Chem. a wide operating range. Pittson. A.