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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 ow-injection system, using the enzyme formaldehyde dehydrogenase and a Os(bpy)2-poly(vinylpyridine) (POs-EA) chemically modied 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 classied 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 xatives; 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 xative 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 Eegriwes, 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

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formaldehyde-assessment methods include visible absorption, HPLC, gas chromatography and uorimetry [11 15]. All these methods require similarly toxic reagents and suffer from a number of interferences, resulting in false positives. Additionally, these methods are impractical for real-time measurements because of the required time for apparatus set-up. Recent efforts have turned toward the development of biological methods of detection combined with physical transducers, biosensors. Electrochemical-based biosensors enable direct, reliable, and reproducible measurements [16,17]. Dehydrogenase-based sensors have attracted attention because of the ubiquity of these enzymes. Some 250 NADH-dependent dehydrogenases and over 150 NADPH-dependent enzymes have been identied [16]. A successful combination between the reactions catalyzed by such enzymes and a transducer might therefore be expected to be of great importance and utility. A general approach, devised for this class of enzymes, would be widely applicable and could provide a basis for enzyme-based electrodes for a variety of analytes, including formaldehyde using formaldehyde dehydrogenase (FDH). Chemical modication of electrode surfaces bestows electrocatalytic properties to the electrode toward NADH electrochemical oxidation [12,13,16 21]. Chemically modied screen-printed electrodes (SPEs) enable the development of a reliable, low-cost, disposable sensor for NADH determination [22,23]. A limited number of devices for the real-time determination of formaldehyde in the gas phase, based on a biosensor using FDH and a chemically modied electrode, has recently been introduced [9,24]. Polymers containing osmium compounds have been used as mediators for NADH electrooxidation in batch reactions, using immobilized glucose dehydrogenase for the determination of glucose [25,26]. In a ow-injection system, a small, precisely metered volume is injected into a owing stream containing the reagent. Further downstream, a ow-through detector monitors the products of the reaction. The reaction products are measured before steady-state conditions are established, and the readout is available within seconds of introducing the sample, so that high sample throughput is possible [27]. Both sample and reagent consumption are low and accuracy and precision are good. Flow-injection systems have proved useful in practical applications in elds as diverse as water and environmental control and agricultural and pharmaceutical analysis [27 31]. In this article we present a novel approach for formaldehyde determination, in aqueous solutions based on the coupling of a biosensor measuring device and a ow-injection system, using immobilized FDH and a Os(bpy)2-poly(vinylpyridine) (POs-EA) modied SPE.

The sensor is designed to measure aqueous solution. For air measurements, as in standard air measurements, the pollutant needs to be transferred from air to an aqueous solution and to be combined with an air sampling device [24,32].

2. Materials and methods

2.1. Materials
NAD+, NADH, FDH [EC 1.2.1.46] from P. putida (specic activity between 3 and 5 U mg 1); alcohol dehydrogenase [EC 1.1.1.1] (ADH) from bakers yeast (specic activity 450 U mg 1); sorbitol dehydrogenase [EC 1.1.1.14] (SDH) from sheep liver (specic activity 6.2 U mg 1); formaldehyde (4% w/v), sorbitol and glycine were purchased from Sigma. Nylon membranes, Immunodyne ABC 5 mm cutoff, were purchased from the Pall Corporation, USA, and used according to the manufacturers instructions. The redox polymerpoly(vinylpyridine) containing complexed (bpy)2OsCl groups and partially quaternized with bromoethylamine, abbreviated POs-EA, was synthesized according to Gregg and Heller [33]. Buffer solutions were prepared immediately before use, using analytical grade K2HPO4 and KH2PO4 purchased from Merck. All other reagents and buffers were of analytical grade. All reagents and electrolyte solutions were prepared using twice distilled water.

2.2. Immunodyne membrane preparation

The FDH enzyme was immobilized onto the Pall Immunodyne membrane (previously cut into 1.2 1.2 cm squares) by dropping aliquots (5 or 10 ml) of enzyme solution (30 mg ml 1 in 0.1 M potassium phosphate buffer pH 8). The membrane was dried in air and then placed for 20 min in a blocking solution containing 0.1 M glycine in 0.1 M potassium phosphate buffer (pH 8). Finally, the membrane was washed in 0.1 M potassium phosphate buffer (pH 8) and kept at 4C, either dry or in the buffer solution, until use.

2.3. Screen -printed electrodes

SPEs were purchased from Gwent Electronic Materials, UK. The electrodes were composed of a carbon working electrode, a carbon auxiliary electrode, and an Ag  AgCl  0.1 M KCl reference electrode. All working solutions contained 0.1 M KCl. The working electrode was modied using the redox polymer POs-EA crosslinked to a commercially available diepoxide poly(ethylene glycol) diglycidyl ether, according to Gregg and Heller [33]. POs-EA solution (25 ml, 4 mg ml 1) was mixed with 5 ml of the diepoxide cross-linking

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agent polyethylene glycol (PEG) (3 mg ml 1). The mixture was thoroughly mixed, and then an aliquot of 5 ml was applied to the working electrode surface. The electrode was left to dry overnight (16 h) at room temperature (r.t.) (20 9 2C) with a cover to protect from dust and light.

2.4. The electrochemical ow cell

The electrochemical system was based on amperometric measurements. The SPE was placed in a home-

made micro (30 ml cell volume) ow cell, connected to a syringe pump and to an injector equipped with a 5 ml injection loop. The enzymatic membrane was placed directly onto the SPE. The electrodes were connected to a computer-controlled BAS 100B potentiostat. The electrochemical cell was washed with the working solution, 0.5 mM NAD+ + 0.1 M KCl + 0.1 M potassium phosphate buffer solution (pH 8), using a 5 ml syringe. A schematic diagram is shown in Fig. 1. The potentiostat, the injector, and the software were purchased from BAS Bioanalytical Systems (BAS, USA).

2.5. Formaldehyde, sorbitol and alcohol determination

Known concentrations of formaldehyde, sorbitol, or ethanol in a solution containing 0.5 mM NAD+ + 0.1 M KCl + 0.1 M potassium phosphate buffer (pH 8), were injected into the cell through the 5 ml injection loop. The ow rate was optimized for each enzyme used. All measurements were performed at r.t. (20 9 2C).

3. Results and discussion The formaldehyde biosensor was based on the following sequence of reactions [9]: HCHO + NAD+ + H2O HCOOH + NADH + H+ (1)
Fig. 1. The electrochemical cell setup. (I) Screen printed electrodes. (II) The electrochemical ow cell. (III) The whole measuring system.

NADH + Osox NAD+ + Osred + H+ Osred Osox + 2e

(2) (3)

Fig. 2. Amperometric response of the SPE to successive additions of NADH. Numbers depict NADH concentrations. 0.1 M potassium phosphate buffer pH 8, 0.1 M KCl, Eapp = 0.35 V, 50 ml min 1 ow rate. ( ) POs-EA modied electrode, ( ) unmodied electrode.

The sensitivity of electrochemical detection depends upon the efciency of the electron transfer from the NADH via the POs-EA mediator to the carbon electrode. Hence, we rst established the optimal conditions of this reaction. Fig. 2 depicts the amperometric biosensor response to successive injections of different concentrations of NADH (5 ml aliquots) into the ow system. The working electrode was held at 350 mV versus Ag  AgCl. Each peak represents the increase in current, due to the oxidation of NADH, as shown in Eq. (3). Fig. 3 shows cyclic voltammograms of the POs-EAmodied electrode, obtained in the ow cell with FDH immobilized on an Immunodyne membrane, in the presence or absence of 0.5 mM formaldehyde. The addition of formaldehyde resulted in a higher anodic current. Fig. 4 shows the response of the biosensor to successive injections of different concentrations of formaldehyde, ranging from 30 ng ml 1 to 4.5 mg ml 1. The rapid response and the high sensitivity are clearly demonstrated. Although tails are observed on the de-

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Fig. 3. Cyclic voltammograms of POs-EA/PEG, modied SPE, in the presence and absence of formaldehyde. 300 mg FDH immobilized on Immunodyne membrane, () Background, ( ) 0.5 mM formaldehyde.

the current response decreased. NAD+ is a known inhibitor of the direct electrochemical oxidation process of NADH [21]; which may also be true for the mediated process, thus requiring further investigation. As has been observed in many studies, the reaction between the mediator and NADH varied with the pH of the contacting buffer in an inverse manner, so that the higher the pH, the lower the reaction rate. This phenomenon could be due to a pH-related change in the osmium mediator orientation on the electrode or to an inherent reaction mechanism [12,21,34,35]. The optimal pH found by us was pH 8.0 (data not shown), and the optimal ow rate was 50 ml min 1. This ow rate allowed enough time for the enzyme to react with both the substrate and the cofactor. The optimal enzyme loading was 300 mg. Reducing the enzyme concentration on the membrane by half, from 300 to 150 mg, did not signicantly change the response. Such, concentrations are similar to literature values [9,24]. At low concentrations of formaldehyde (30 ng ml 1 to 1.5 mg ml 1) injected into the electrochemical cell, the current response showed a linear relation (y = 15.8 + 0.06x, R 2 = 0.997). As expected for an enzymatic reaction the linear relation does not hold at high formaldehyde concentrations. A Lineweaver Burk plot resulted in a straight line with R 2 = 0.994 and the Km calculated from the plot was 1.74 mg ml 1 (58 mM). This value agrees with the formaldehyde data in the literature: 90 and 56 mM [9,24]. The stability of the immobilized enzyme on the Immunodyne membrane is shown in Fig. 6. When the membranes were stored at 4C in potassium phosphate buffer, pH 8, the response was stable and linear over 7

Fig. 4. Detection of formaldehyde. Amperometric response of the sensor to injections of formaldehyde. 0.1 M potassium phosphate buffer pH 8. Eapp = 0.35 V. Numbers depict formaldehyde concentrations.

scending portion of the signal current, the response to formaldehyde is reproducible and linear. These tails could be due to air bubbles in the probably not ideal, homemade, inexpensive and portable ow cell. In control experiments without FDH, no response to formaldehyde injection was observed. We further optimized the formaldehyde sensor performance by optimizing the cofactor concentration, pH, ow rate and enzyme concentration. Fig. 5 shows the dependence of the current response on the cofactor concentration. The optimal NAD+ concentration found was 0.5 mM. At higher concentrations of NAD+

Fig. 5. Effect of NAD+ concentration on the sensor response. Enzyme loading 300 mg, 0.1 M potassium phosphate buffer pH 8, Eapp = 0.35 V. Dotted columns: 0.3 mg ml 1 formaldehyde, horizontal lines columns: 0.6 mg ml 1 formaldehyde, diagonal lines columns: 1.5 mg ml 1 formaldehyde.

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Fig. 6. Long-term stability of FDH immobilized on Immunodyne membrane. , Dry membrane, day 1; , wet membrane, day 1; , dry membrane, day 7; , wet membrane, day 7; 2, wet membrane, day 30. 0.1 M pH 8, 0.5 mM NAD+, Eapp = 0.35 V, enzyme loading 300 mg.

days. After 30 days, however, only 6% of the initial enzymatic activity remained. Dried membranes stored sealed with silica gel at 4C showed a linear response over a period of 7 days, however, 30% of the activity was lost. Dry storage of the immobilized membrane at r.t. resulted in a complete loss of bound enzyme activity and/or fouling of the membrane (data not shown). The simplicity of this system enables the quick preparation of an immobilized membrane, which can retain its bound enzyme activity for over a week and thus is ideally suited for on-site measurements. Methanol is often used as a stabilizer for formaldehyde solutions and can also be found in mixed-waste vapors containing formaldehyde [36]. In fact, commercial formalin contains methanol. We therefore examined the response of the sensor to methanol and found that the sensor retained its specicity for formaldehyde and did not respond to equivalent additions of methanol (data not shown). 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 [12,16,22,35]. The same design can be used with other dehydrogenase enzymes. Immobilized ADH, shown in Fig. 7(A), can be used for developing an ethanol biosensor [18,21], which is pertinent to fermentation. The successive addition of ethanol, at increasing concentrations ranging from 46 ng ml 1 to 7.8 mg ml 1, resulted in a parallel successive increase in current response. At concentrations ranging from 46 ng ml 1 to 2.3 mg ml 1 (y = 0.74 + 2.9x, R 2 = 0.996), a linear correlation between concentration and response

Fig. 7. Detection of sorbitol and ethanol. (A) Amperometric response to successive ethanol injections. (B) Amperometric response to successive sorbitol injections. 0.5 mM NAD+ + 0.1 M potassium phosphate buffer pH 8. Eapp = 0.35 V. Numbers depict substrate concentration in mg ml 1.

was achieved. This biosensor will be able to detect minute (10 9 g) amounts of alcohol using a small enzyme loading of 25 mg. Fig. 7(B) depicts the response of SDH, immobilized on the Immunodyne membrane at 300 mg enzyme loading, to successive sorbitol additions, at concentrations ranging from 0.46 to 4.6 mg ml 1. The correlation between the increasing concentration of sorbitol and the current response resulted in a linear calibration curve (y = 7.6 + 7.5x, R 2 = 0.992). The data demonstrate the possibility of using such biosensors for the measurement of sorbitol, an important chemical in the food industry.

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4. Conclusions In this work, we have presented a generally applicable approach and three examples that are based on the mediated oxidation of enzymatically produced NADH. In our experiments, three dehydrogenases were used, formaldehyde dehydrogenase, ADH and SDH for the respective detection of formaldehyde, alcohol and sorbitol, emphasizing the formaldehyde detection. A novel combination of a POs-EA modied SPE, an immobilized enzyme on an Immunodyne membrane, and a micro ow-injection system allowed us to detect formaldehyde with the high sensitivity and selectivity that is required by the EPA and OSHA. The detection limit of the sensor was 30 ng ml 1 in the solution corresponding to 0.2 vppb concentration of the formaldehyde in the space above the formaldehyde solution [9]. This detection limit is the lowest ever reported for formaldehyde detection using electrochemical and enzymatic methods [9,19,24,36,37]. The sensor response was linear over a wide range of concentrations (30 ng ml 1 to 1.5 mg ml 1). At higher concentrations, the response was not linear but the sensor remained sensitive to increasing amounts of the substrate. Despite the presence of methanol in commercial formalin solutions, the formaldehyde-selective sensor did not respond to injections of methanol. The enzyme-modied membranes could be stored for 7 days at 4C without losing activity. Incorporating the sensor into the micro-owinjection system enabled us to use very small amounts (sub-nanogram) of sample. Such small amounts are notably important when dealing with toxic chemicals. To conclude, the sensor described in this work assembles the basic requirements for an on-the-spot biosensor: simplicity, selectivity, sensitivity, together with reusability, a wide operating range, and low cost.
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