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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 toelectrochemistry

Abstract

This paper reports the development of a novel detection method, based on the coupling of a biosensor measuring device anda flow-injection system, using the enzyme formaldehyde dehydrogenase and a Os(bpy)2-poly(vinylpyridine) (POs-EA) chemicallymodified screen-printed electrode. The sensor can detect 30 ng ml−1 of formaldehyde in aqueous solution (corresponding tosub-ppb atmospheric concentrations of formaldehyde). The sensor is selective, inexpensive, stable over several days, anddisposable, as well as simple to manufacture and operate. The system described here can easily be adapted to other substratesusing 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 unprecedentedamount of hazardous air pollution (HAP) has beencreated. Currently, the largest source of HAP is auto-motive engine exhaust, which contains several toxicpollutants, such as formaldehyde. Formaldehyde, awidely used industrial chemical in many manufacturingprocesses, is toxic, allergenic and accumulates in the airover cities. Formaldehyde has been classified as a hu-man carcinogen by both the US Environmental Protec-tion Agency (EPA) and the World Health Organization[1,2]. Formaldehyde is released as a by-product ofincomplete hydrocarbon combustion and is emitted at arate of 700 mg l−1 gasoline [3]. Approximately 4×1011

kg formaldehyde is formed in the troposphere annuallythrough the photochemical oxidation of released hydro-carbons [2]. Most commercial formaldehyde is pro-duced from methanol for use in many industrialprocesses, including wood fixatives; dry cleaning solu-tions; solvent use; boiler use; chemical production; oil,gas, and petroleum production, as well as paper andpulp production; the cosmetics industry and the textile

industry [1,2,4,5]. In hospitals, formaldehyde is used asa disinfectant, as well as a fixative by pathologists,medical technicians, and researchers. Today, approxi-mately 10 megatons per year of formaldehyde are pro-duced [6].

Formaldehyde accumulates in the atmosphere overcities and is known to induce asthma-like symptoms inhumans [7]. Polluted urban air contains between 0.010and 0.160 ppm formaldehyde; in urban air on a sunnyday, formaldehyde has a half-life of �50 min [8].Exposure to formaldehyde can cause central nervoussystem damage; blood, immune system and develop-mental disorders; as well as blindness and respiratorydisease [3,9,10]. Vapor-phase formaldehyde is linked tonasophrayngaeal carcinomas in rats.

Standards have been set by the Occupational Safetyand Health Administration, as well as by the EPA, tolimit human exposure and health risk in occupationsinvolving formaldehyde use. Formaldehyde levels mustbe accurately monitored to comply with these stan-dards. Colorimetric detection methods, such asDeniges’ method, and Eegriwe’s, have been knownsince the beginning of the 20th century [5]. Unfortu-nately, these methods, reagents and reaction productsare often just as harmful to human health and theenvironment as is formaldehyde. Currently, standard

* Corresponding author. Tel.: +972-3-6409836; fax: +972-3-6409407.

0022-0728/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S 0022 -0728 (00 )00170 -4

Y. Herschko6itz et al. / Journal of Electroanalytical Chemistry 491 (2000) 182–187 183

formaldehyde-assessment methods include visible ab-sorption, HPLC, gas chromatography and fluorimetry[11–15]. All these methods require similarly toxicreagents and suffer from a number of interferences,resulting in false positives. Additionally, these methodsare impractical for real-time measurements because ofthe required time for apparatus set-up. Recent effortshave turned toward the development of biologicalmethods of detection combined with physical transduc-ers, biosensors. Electrochemical-based biosensors en-able direct, reliable, and reproducible measurements[16,17].

Dehydrogenase-based sensors have attracted atten-tion because of the ubiquity of these enzymes. Some250 NADH-dependent dehydrogenases and over 150NADPH-dependent enzymes have been identified [16].A successful combination between the reactions cata-lyzed by such enzymes and a transducer might thereforebe expected to be of great importance and utility. Ageneral approach, devised for this class of enzymes,would be widely applicable and could provide a basisfor enzyme-based electrodes for a variety of analytes,including formaldehyde using formaldehyde dehydroge-nase (FDH).

Chemical modification of electrode surfaces bestowselectrocatalytic properties to the electrode towardNADH electrochemical oxidation [12,13,16–21]. Chem-ically modified screen-printed electrodes (SPEs) enablethe development of a reliable, low-cost, disposable sen-sor for NADH determination [22,23]. A limited numberof devices for the real-time determination of formalde-hyde in the gas phase, based on a biosensor using FDHand a chemically modified electrode, has recently beenintroduced [9,24]. Polymers containing osmium com-pounds have been used as mediators for NADH elec-trooxidation in batch reactions, using immobilizedglucose dehydrogenase for the determination of glucose[25,26].

In a flow-injection system, a small, precisely meteredvolume is ‘injected’ into a flowing stream containing thereagent. Further downstream, a flow-through detectormonitors the products of the reaction. The reactionproducts are measured before ‘steady-state’ conditionsare established, and the readout is available withinseconds of introducing the sample, so that high samplethroughput is possible [27]. Both sample and reagentconsumption are low and accuracy and precision aregood. Flow-injection systems have proved useful inpractical applications in fields as diverse as water andenvironmental control and agricultural and pharmaceu-tical analysis [27–31].

In this article we present a novel approach for form-aldehyde determination, in aqueous solutions based onthe coupling of a biosensor measuring device and aflow-injection system, using immobilized FDH and aOs(bpy)2-poly(vinylpyridine) (POs-EA) modified 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 anaqueous solution and to be combined with an airsampling device [24,32].

2. Materials and methods

2.1. Materials

NAD+, NADH, FDH [EC 1.2.1.46] from P. putida(specific activity between 3 and 5 U mg−1); alcoholdehydrogenase [EC 1.1.1.1] (ADH) from bakers yeast(specific activity 450 U mg−1); sorbitol dehydrogenase[EC 1.1.1.14] (SDH) from sheep liver (specific activity6.2 U mg−1); formaldehyde (4% w/v), sorbitol andglycine were purchased from Sigma. Nylon membranes,Immunodyne® ABC 5 mm cutoff, were purchased fromthe Pall Corporation, USA, and used according to themanufacturer’s instructions. The redox polymer-poly(vinylpyridine) containing complexed (bpy)2OsClgroups and partially quaternized with bromoethy-lamine, abbreviated POs-EA, was synthesized accordingto Gregg and Heller [33]. Buffer solutions were pre-pared immediately before use, using analytical gradeK2HPO4 and KH2PO4 purchased from Merck.

All other reagents and buffers were of analyticalgrade. All reagents and electrolyte solutions were pre-pared using twice distilled water.

2.2. Immunodyne® membrane preparation

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

2.3. Screen-printed electrodes

SPEs were purchased from Gwent Electronic Materi-als, UK. The electrodes were composed of a carbonworking electrode, a carbon auxiliary electrode, and anAg � AgCl � 0.1 M KCl reference electrode. All workingsolutions contained 0.1 M KCl. The working electrodewas modified using the redox polymer POs-EA cross-linked to a commercially available diepoxide poly-(ethylene glycol) diglycidyl ether, according to Greggand 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). Themixture was thoroughly mixed, and then an aliquot of5 ml was applied to the working electrode surface. Theelectrode was left to dry overnight (16 h) at roomtemperature (r.t.) (2092°C) with a cover to protectfrom dust and light.

2.4. The electrochemical flow cell

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

made micro (30 ml cell volume) flow cell, connected toa syringe pump and to an injector equipped with a 5 mlinjection loop. The enzymatic membrane was placeddirectly onto the SPE. The electrodes were connected toa computer-controlled BAS 100B potentiostat. Theelectrochemical cell was washed with the working solu-tion, 0.5 mM NAD++0.1 M KCl+0.1 M potassiumphosphate buffer solution (pH 8), using a 5 ml syringe.A schematic diagram is shown in Fig. 1.

The potentiostat, the injector, and the software werepurchased from BAS Bioanalytical Systems (BAS,USA).

2.5. Formaldehyde, sorbitol and alcohol determination

Known concentrations of formaldehyde, sorbitol, orethanol in a solution containing 0.5 mM NAD++0.1M KCl+0.1 M potassium phosphate buffer (pH 8),were injected into the cell through the 5 ml injectionloop. The flow rate was optimized for each enzymeused. All measurements were performed at r.t. (2092°C).

3. Results and discussion

The formaldehyde biosensor was based on the fol-lowing sequence of reactions [9]:

HCHO+NAD++H2O�HCOOH+NADH+H+

(1)

NADH+Osox�NAD++Osred+H+ (2)

Osred�Osox+2e− (3)

The sensitivity of electrochemical detection dependsupon the efficiency of the electron transfer from theNADH via the POs-EA mediator to the carbon elec-trode. Hence, we first established the optimal condi-tions of this reaction. Fig. 2 depicts the amperometricbiosensor response to successive injections of differentconcentrations of NADH (5 ml aliquots) into the flowsystem. The working electrode was held at 350 mVversus Ag � AgCl. Each peak represents the increase incurrent, due to the oxidation of NADH, as shown inEq. (3).

Fig. 3 shows cyclic voltammograms of the POs-EA-modified electrode, obtained in the flow cell with FDHimmobilized on an Immunodyne® membrane, in thepresence or absence of 0.5 mM formaldehyde. Theaddition of formaldehyde resulted in a higher anodiccurrent.

Fig. 4 shows the response of the biosensor to succes-sive injections of different concentrations of formalde-hyde, ranging from 30 ng ml−1 to 4.5 mg ml−1. Therapid response and the high sensitivity are clearlydemonstrated. Although tails are observed on the de-

Fig. 1. The electrochemical cell setup. (I) Screen printed electrodes.(II) The electrochemical flow cell. (III) The whole measuring system.

Fig. 2. Amperometric response of the SPE to successive additions ofNADH. Numbers depict NADH concentrations. 0.1 M potassiumphosphate buffer pH 8, 0.1 M KCl, Eapp=0.35 V, 50 ml min−1 flowrate. (—) POs-EA modified electrode, (– – –) unmodified electrode.

Y. Herschko6itz et al. / Journal of Electroanalytical Chemistry 491 (2000) 182–187 185

Fig. 3. Cyclic voltammograms of POs-EA/PEG, modified SPE, in thepresence and absence of formaldehyde. 300 mg FDH immobilized onImmunodyne® membrane, (—) Background, (– – –) 0.5 mM formal-dehyde.

the current response decreased. NAD+ is a knowninhibitor of the direct electrochemical oxidation processof NADH [21]; which may also be true for the medi-ated process, thus requiring further investigation. Ashas been observed in many studies, the reaction be-tween the mediator and NADH varied with the pH ofthe contacting buffer in an inverse manner, so that thehigher the pH, the lower the reaction rate. This phe-nomenon could be due to a pH-related change in theosmium mediator orientation on the electrode or to aninherent reaction mechanism [12,21,34,35]. The optimalpH found by us was pH 8.0 (data not shown), and theoptimal flow rate was 50 ml min−1. This flow rateallowed enough time for the enzyme to react with boththe substrate and the cofactor. The optimal enzymeloading was 300 mg. Reducing the enzyme concentra-tion on the membrane by half, from 300 to 150 mg, didnot significantly change the response. Such, concentra-tions 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, R2=0.997). As expected for an enzymaticreaction the linear relation does not hold at high form-aldehyde concentrations. A Lineweaver–Burk plot re-sulted in a straight line with R2=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 theliterature: 90 and 56 mM [9,24].

The stability of the immobilized enzyme on the Im-munodyne® membrane is shown in Fig. 6. When themembranes were stored at 4°C in potassium phosphatebuffer, pH 8, the response was stable and linear over 7

Fig. 4. Detection of formaldehyde. Amperometric response of thesensor to injections of formaldehyde. 0.1 M potassium phosphatebuffer pH 8. Eapp=0.35 V. Numbers depict formaldehyde concentra-tions.

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, horizon-tal lines columns: 0.6 mg ml−1 formaldehyde, diagonal lines columns:1.5 mg ml−1 formaldehyde.

scending portion of the signal current, the response toformaldehyde is reproducible and linear. These tailscould be due to air bubbles in the probably not ideal,homemade, inexpensive and portable flow cell. In con-trol experiments without FDH, no response to formal-dehyde injection was observed.

We further optimized the formaldehyde sensor per-formance by optimizing the cofactor concentration, pH,flow rate and enzyme concentration. Fig. 5 shows thedependence of the current response on the cofactorconcentration. The optimal NAD+ concentrationfound was 0.5 mM. At higher concentrations of NAD+

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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 loading300 mg.

Fig. 7. Detection of sorbitol and ethanol. (A) Amperometric responseto successive ethanol injections. (B) Amperometric response to succes-sive sorbitol injections. 0.5 mM NAD++0.1 M potassium phosphatebuffer pH 8. Eapp=0.35 V. Numbers depict substrate concentrationin mg ml−1.

days. After 30 days, however, only 6% of the initialenzymatic activity remained. Dried membranes storedsealed with silica gel at 4°C showed a linear responseover a period of 7 days, however, 30% of the activitywas lost. Dry storage of the immobilized membrane atr.t. resulted in a complete loss of bound enzyme activityand/or fouling of the membrane (data not shown). Thesimplicity of this system enables the quick preparationof an immobilized membrane, which can retain itsbound enzyme activity for over a week and thus isideally suited for on-site measurements.

Methanol is often used as a stabilizer for formalde-hyde solutions and can also be found in mixed-wastevapors containing formaldehyde [36]. In fact, commer-cial formalin contains methanol. We therefore exam-ined the response of the sensor to methanol and foundthat the sensor retained its specificity for formaldehydeand did not respond to equivalent additions ofmethanol (data not shown).

Coupling the electrocatalytic oxidation of NADH bythe mediator to the reaction catalyzed by the NAD+-dependent dehydrogenase enzymes enables the con-struction of amperometric biosensors for a large varietyof other substrates [12,16,22,35]. The same design canbe used with other dehydrogenase enzymes. Immobi-lized ADH, shown in Fig. 7(A), can be used for devel-oping an ethanol biosensor [18,21], which is pertinentto fermentation. The successive addition of ethanol, atincreasing concentrations ranging from 46 ng ml−1 to7.8 mg ml−1, resulted in a parallel successive increase incurrent response. At concentrations ranging from 46 ngml−1 to 2.3 mg ml−1 (y=0.74+2.9x, R2=0.996), alinear correlation between concentration and response

was achieved. This biosensor will be able to detectminute (10−9 g) amounts of alcohol using a smallenzyme loading of 25 mg.

Fig. 7(B) depicts the response of SDH, immobilizedon the Immunodyne® membrane at 300 mg enzymeloading, to successive sorbitol additions, at concentra-tions ranging from 0.46 to 4.6 mg ml−1. The correlationbetween the increasing concentration of sorbitol andthe current response resulted in a linear calibrationcurve (y=7.6+7.5x, R2=0.992). The data demon-strate the possibility of using such biosensors for themeasurement of sorbitol, an important chemical in thefood industry.

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4. Conclusions

In this work, we have presented a generally applica-ble approach and three examples that are based on themediated oxidation of enzymatically produced NADH.In our experiments, three dehydrogenases were used,formaldehyde dehydrogenase, ADH and SDH for therespective detection of formaldehyde, alcohol and sor-bitol, emphasizing the formaldehyde detection.

A novel combination of a POs-EA modified SPE, animmobilized enzyme on an Immunodyne® membrane,and a micro flow-injection system allowed us to detectformaldehyde with the high sensitivity and selectivitythat is required by the EPA and OSHA. The detectionlimit of the sensor was 30 ng ml−1 in the solutioncorresponding to 0.2 vppb concentration of the formal-dehyde in the space above the formaldehyde solution[9]. This detection limit is the lowest ever reported forformaldehyde detection using electrochemical and enzy-matic methods [9,19,24,36,37]. The sensor response waslinear over a wide range of concentrations (30 ng ml−1

to 1.5 mg ml−1). At higher concentrations, the responsewas not linear but the sensor remained sensitive toincreasing amounts of the substrate. Despite the pres-ence of methanol in commercial formalin solutions, theformaldehyde-selective sensor did not respond to injec-tions of methanol. The enzyme-modified membranescould be stored for 7 days at 4°C without losingactivity. Incorporating the sensor into the micro-flow-injection system enabled us to use very small amounts(sub-nanogram) of sample. Such small amounts arenotably important when dealing with toxic chemicals.

To conclude, the sensor described in this work assem-bles the basic requirements for an on-the-spot biosen-sor: simplicity, selectivity, sensitivity, together withreusability, a wide operating range, and low cost.

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