233 z Flow Injection Amperometric Detection of Ammonia Using a Polypyrrole-Modified Electrode and Its Application in Urea and Creatinine Biosensors zyxwv Marek Trojanowicz,*' Andrzej Lewenstam, Szczepek Tadeusz Krawczyriski Vel Krawczyk, zyxw ' Ilkka Lahdesmaki, + and Wojciech + Department of Chemistry, University of Warsaw, Pasteura I, PL-02-093 Warsaw, Poland Department of Analytical Chemistry, Abo Akademi University, FI-20500 Turku, Finland Pharmaceutical Research Institute, Rydygiera 8, PL-01-793 Warsaw, Poland zyxwvu +t + I + Received: January 12, 1995 Final version: March 27, 1995 Abstract The interaction of unprotonated ammonia molecules with polypyrrole (PPy) is employed for the amperometric detection of ammonia with the use of an anodically polarized platinum electrode modified with PPy as the working electrode. The polymer was obtained by electropolymerization in potentiostatic conditions from aqueous solutions. Chloride was found to be the most suitable dopant for PPy from several dopants studied. Satisfactory dynamic characteristics of detection permit its application to the determination of ammonia in a flow injection system up to 100 pM of analyte with a detection limit of 0.6 pM. Such a detector of ammonia was also employed as the internal detector in amperometric biosensors for urea and creatinine by the immoblizdtion in a different way at the PPy surface of urease and creatinine iminohydrolase, respectively. An amperometric biosensor of very low sensitivity was obtained by urease entrapment in the PPy layer during the electropolymerization. The urea membrane biosensor was successfully applied in the determination of urea in human blood samples. On the basis of results of elemental analysis, UV and IR spectroscopy some suggestions are presented about the mechanism of the interaction between PPy and ammonia and about the anodic electrode process occurring in the amperometric detection of ammonia. Keywords: Ammonia, Polypyrrole, Urea, Creatinine, Flow injection analysis 1. Introduction The first report about the possibility of the use of a polypyrrole (PPy)-modified electrode for ammonia detection was published by Kanazawa et al. [l], who reported that exposing PPy to ammonia gas decreases the room tempera- ture conductivity of PPy by a factor of 10. The authors of that work relate that ammonia interacts only weakly with PPy and can be readily pumped off restoring the conductvity to the original value. The sensitivity of the resistance of the PPy film to ammonia was demonstrated by Nylander et al. [2]. Gustafsson zyxwvuts et al. 131 found that a low concentration of ammonia produced a reversible increase in the resistance of PPy, while concentra- tions of ammonia greater than zyxwvutsr 1 atm, or the presence of water, led to a permanent increase in resistance. That increase was attributed to nucleophilic attack of ammonia or hydroxide on the polymer leading to loss of conjugation and even ring opening. It has been reported that electrochemically prepared PPy film could detect 0.01% ammonia 141. Also an ultrathin layer of PPy coated on nonconducting substrates can be used as a chemiresistor for the sensitive detection of ammonia and hydrazine zyxwvutsrq [5]. The reversed changes of resistance were observed in d.c. con- ductivity measurements performed on PPy films doped with hexa- cyanoferrate(II), where some response to gaseous ammonia at high concentrations was found [6]. It was concluded that ammonia acts as a reversible dedopant, causing a decrease of conductivity. From the study involving measurements of the PPy resistance change and mass changes in the presence of selected gases and vapors, including ammonia, it was concluded that the response mechanism of PPy sensitivity is due to a mixed response involving electronic effects and physical effects 171. Mass changes of PPy were also utilized for the design of piezoelectric ammonia gas sensors with PPy deposited electrochemically on the surface of the gold electrode of a piezoelectric detector [7,8]. We have already demonstrated that a PPy-modified platinum electrode can be successfully used as an amperometric detector of ammonia in aqueous solutions [9]. The first application of the detection of ammonia with a PPy-modified electrode for the design of an amperometric urea biosensor was reported with the use of the air-gap ammonia gas microelectrode [lo]. Amperometric sensitivity to ammonia in aqueous solutions was applied in flow injection determination of urea in the system with flow-through enzyme reactor [9]. Recently, the potentiometric response to urea was reported for the electrode with urease entrapped in the PPy layer with a potential change of 17.5 mV per decade within 0.5 to 500 mM, however, with substantially decreased sensitivity in the presence of potassium ions [I 1,121. This method of urease immoblization was also applied in obtaining the urea biosensor for which dependence of conductance, capacitance and admittance on urea concentration in aqueous solutions up to 12 mM was observed [13]. No data about the stability and selectivity of such a sensor were reported. As numerous isolated enzymes which degrade various sub- strates to ammonia are available [14], the search for an effective internal detector of ammonia for the design of biosensors is of great importance. The nonactin based ammonium ion-selective electrode most often used for this purpose exhibits insufficient selectivity in the presence of alkali metal ions. Both potentio- metric [14a] and amperometric [I 51 measurements of ammonium nitrogen with ionophore containing membranes require the use of gas permeable membranes to eliminate the alkali metal ion interference. Hence, this study was undertaken on the further optimization of amperometric detection of ammonia with a PPy- modified electrode and on the possibility of the use of such a sensor for the design of amperometric biosensors for substrates decomposed enzymatically to ammonia. Elrc.irounalysis 1996, 8, No. 3 zyxwvutsrqpon 0 VCH Vrrlugsgesellschuft mbH. 0-69469 Weinheim. I996 l040-0397196/0303-233 $ l0.00+.25/0