Amperometric ammonium ion sensor and its application to biosensors

Amperometric ammonium ion sensor and its application to biosensors and Actuators B, 13-14 Amperometric biosensors (1993) 57 57-60 ammonium ion sensor and its application to Yukitaka Yamamoto KjwoMunici...

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Actuators B, 13-14

Amperometric biosensors




ammonium ion sensor and its application to

Yukitaka Yamamoto KjwoMunicipal Junior College of Nursing M&u-Hipshi-takaia-cho, K’ Mitsugi Senda* Lkpomnent of Bio.~cience, Fukui Ref~twal

604 (Japan)

Vniversity,Maracoka-cho,Fukui 910-U (lapan)

Abstract A urea biosensor based on the amperometric determination of ammonia produced by hydrolytic decomposition of urea by urease is descnid. The urease is immobiliied on the ampemmetric ammonium ion-selective electrode sensor, This urea sensor gives the current response proportional to the concentration of urea in the range, of a few to 100 PM in test solutions. The response can be axrected for the residual ammonium ions. The urea sensor was successfullyapplied to the analysis of biological fluids. An amperomchic creatinine biosensor is also de&id.


Biosensors based on potentiometrlc ion sensors using field-effect transistors as well as ion-selective electrodes have been extensively studied and well developed [l, 21. In contrast, this study is concerned with the amperometric (or voltammetric) ion sensors and biosensors based on them. Recent electrochemical studies on the ion transfer at the interface between two immiscible electrolyte solutions (ITIES) or the oil/water (o/w) interface have shown that the o/w interface can be electrochemically polarized and that the transfer of ions that takes place at the polarized potential range across the interface can be studied using polarographic or voltammetric techniques [3-51. Thus, the polarized o/w (e.g. nitrobenzene (NB)hater (W)) interface can function as the ion-selective electrode (ISE) interface which responds to a specified ion that is selectively transferable across the interface. The presence of an ionophore (L, see Fig. 1 below) that selectively associates with a specified ion (e.g. NH,+ in Fig. 1) in the oil or organic phase (e.g., nitrobenzene in Fig. 1) will give the interface the ion selectivity for the specified ion. In previous papers [6,7] an amperometric ammonium ion sensor based on the stated principle has been proposed and was successfully applied to the determination of ammonia in fish meats. Furthermore, an amperometric urea biosensor based on the ampero*Author to whom correspondence should be addressed.


metric determination of ammonia produced by hydra lytic decomposition of urea has also been proposed [8]. In this paper further development of the amperometric urea sensor and its application to the analysis of biological fluids are reported. An amperometric creatinine sensor based on a similar principle is also de&bed.

The principle of the amperometric urea sensor is shown in Fig. 1 and the electrochemical cell for determination of urea concentration in a test solution is represented by 0.M TBAC


CCUA In this cell TBACl, TBATPB and DBl8C6 are tetrabutylammonium chloride, tetrabutylammonium tetraphenylborate and dibenzo-18-crown-6, respectively, and the mark * indicates the polarizable interface (that is, the ammonium ion-selective electrode interface). The amperometric ammonium ion-selective electrode or the

Q 1993

- Elsevier Sequoia. Au rights reserved



GPM urease


( PH

N Hr.-L



test solution


( pH 9.0 1



I/ N HI’

l+ co2

urea L

t Fig. 1. Principle of the urea sensor. A

icm ammonia Sensor is essentially the same as that described previously [7j. The polarized NB(DBNC6)IW interface was stabilized by placing a hydrophilic semipermeable membrdne (HSM, a dialysis membrane of 20 pm thickness, Visking Co.) at the interface and the HSM-coated interface was further covered by a gas permeable membrane (GPM, a Teflon membrane of 50 pm thickness, Sumitomo Denko K.K. FP-200) with an inner solution of 0.05 M MgCl, and 0.05 M L-lysine with a nylon mesh spacer between the HSM and GPM. Also, the inner solution was connected to the right hand Ag/ AgCl reference electrode. The GPM gives the electrode a high selectivity for the ammonium ion but with a prolonged response time. Interference of sodium and potassium ions (and other non-volatile ions) is eliminated. A solution of urease (usually, of 100 U quantities in 0.1 M tris-HCl pH 8.5 buffer containing 15% bovine serum albumin) was placed on the covering GPM, the solvent was evaporated and the surface of the ureaseplaced, GPM-covered ammonia sensor was further covered by an HSM with a spacer (also made of an HSM) to immobilize urease on the ammonia sensor. The structure of the urea sensor is illustrated in Fig. 2. The creatinine sensor was fabricated by much the same way but using creatinine deiminase (50 U) in place of urease.

Electrochemical measurements The HSM-covered enzyme-immobilized sensor was

immersed in 0.1 M tri.+HCl buffer of pH 8.5 for 2 h before use then immersed in test solution (see cell A) to follow the current response. The pulse amperometry technique was used to determine the current response of the sensor; a voltage pulse of 100 ms duration and of constant amplitude giving the limiting current, e.g. 0.2 V with the initial potential of 0.25 V for ammonium ion with cell A, was applied every 5 s to the electrode interface. Details of the electrochemical measurements are described elsewhere [9].




Fig. 2. Schematic cross section of a laboratory-made

urea sensor.


Urease (jack bean, Grade II, 160 U/mg, Toyobo Co.) and creatinine deiminase (EC, 3,5,4,21, microorganism, Sigma Chemical Co.) were purchased and used as received. The other chemicals are described elsewhere [7, 81.

Results and discussion Figure 3 shows the pulse amperometric response of a urea sensor for successive addition of urea to the test solution under stirring. The response time of the sensor was about 300 s (to reach 90% of steady state) for laboratory-made sensors with immobilized urease more than 100 U. With decreasing amounts of the immobilized urease of less than 100 U the response time tended to increase (data not shown). The calibration curve was linear in the range from a few to 100 PM of urea in the test solution. The relative standard deviation of the current response was 3.8% (n=5, at 20 PM) and the lifetime was more than 20 days. The amperometric sensor gives a current response which is proportional to the concentration of analyte, whereas the potentiometric sensor gives a potential response which changes linearly with the logarithm of concentration (activity) of the analyte. One of the advantages of the amperometric sensor is that the correction for the residual current, e.g. due to the residual ammonium ion in the determination of urea, in the test solution can easily be done. For this purpose


Fig. 3. Pulse amperometric response (A) of a urea sensor for successive addition of urea at the time indicated by the arrows (7 ), followed by washing of the sensor with 0.1 M his-HCI buffer @H 8.5) at the time indicated by the downward arrow (1). Initial potential: 0.25 V, applied potential pulse: 0.2 V height and 100 ms width at 5 s interval. B: base current; C: A-B.

a (ammonium ion) sensor of the same structure as the urea sensor (Fig. 2 and cell A) but urease being removed from the enzyme-immobilized layer was fabricated. This urease-removed urea sensor gave a current response proportional to the concentration of ammonium ion but no response to urea in the test solution (see the closed circles in Fig. 4). Furthermore, the current sensitivity of this urease-removed urea sensor to the ammonium ion coincided with that of the (normal) urea sensor within experimental error, as seen by the agreement between the current responses (open and closed circles) of the two sensors to 80 NM ammonium ion in the test solution in Fig. 4. Generally, the reproducibility of the laboratory-made urease-removed as well as normal urea sensors with respect to the current sensitivity to ammonium ion was f 5%; whereas the variation in the current sensitivity of the laboratorymade (normal) urea sensors was large, in some cases up to 50%. Correction, if necessary, for the current sensitivity by use of the calibration curve is feasible. Thus, the urease-removed urea sensor gives the current response proportional to the concentration of the residual ammonium ion while the (normal) urea sensor gives the current response which is the sum of the current responses each proportional to the concentrations of ammonium ion and urea (open circles in Fig. 4) in test solutions. Subtraction of the former from the latter gives the concentration of urea corrected for the (residual) ammonium ion concentration. Table 1 shows the analytical results of urea-nitrogen contents in sera and saliva obtained with the laboratotymade urea sensor in comparison with those by the



4 a .



1 uH

Fig. 4. Calibration curves (current responses) of the ureaseremoved urea sensor (closed circles) and the (normal) urea sensor (open circles) in solutions containing both urea and ammonium ion at 80+0, 40+20, 40+40, 20+60 and 0+80 PM.

60 TABLE 1. Urea-nitrogen (UN) contents in sera and saliva determined by the amperometric urea sensor



This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

Animal serum (Pathonorm H) Aninlalserum (Pathonorm L) Human serum Saliva


UN (mg/lOO ml) Biosensor







x200 Xlocl

14.6 8.1

References 8.2(6.5-9.0) 14.0

‘The certified value of the standard reference materials.

urease-indophenol (spectrophotometric) method [lo]. The results indicate that the amperometric urea sensor can be used for the analysis of biological fluids. The creatinine sensor also showed similar characteristics to those of the urea sensor and gave a current response proportional to the concentration of creatinine in test solutions in the range O-140 fl [ll].

Electrochemical biosensom can be constructed on the basis of the amperometric (or voltammetric) ionselective electrode sensor and are applicable to the analysis of biological and other samples. The fabrication of disposable sensors appears promising.

1 A. P. F. Turner, I. Kambe and G. Wilson (eds.), Biorensoar, Fudamentah and Applicatkms,Oxford University Press, Oxford, 1987, pp. 770. 2 D. L. Wise, Bioinshmentation, Reseanzh, Development and Applicatiotq Butterworths, Boston, MA, 1990, pp. 1563. 3 J. Koryta, Electrochemical polarization phenomena at the interface of hvo immiscible electrolyte solutions-I, II, III, Ekximchim Acta, 24 (1979) 293-300,27 (1984) 445-452; 33 (1989) 189-197. 4 H. H. J. Girault and D. J. Schiliriu, Electrochemistry of liquid-liquid interfaces, Eleebvanal. Ch., 15 (1989) I-140. 5 M. Senda, T. Kakiuchi and T. Osakai, Electrochemistry at the interface between two hmniscible electrolyte solutions, Electmchin~ Acta, 36 (1991) 253-262. 6 T. Osakai, T. Kakutani and M. Sends, A novel amperometric ammonia sensor, Anal. Sci., 3 (1987) 521-526. 7 Y. Yamamoto, T. Nuno, T. Osakai and M. Senda, A volatile amine sensor based on the amparometric ion-selective electrode, Bun& i&&u, 38 (1989) 589-595. 8 T. Osakai, T. Kakutani and M. Senda, A novel amperometric urea sensor, Anal. Sci, 4 (1988) 529-530. 9 T. Osakai, T. Nuno, Y. Yamamoto and M. Senda, A microcomputer-contmlled system for ion-transfer vohammetry, Bun&i k&d.z~, 38 (1989) 479-W. 10 S. Kanai. Rinsw Kensa-Ho Tziw (Handbook of Clinical Ana&rF),’ Kin&a Syuppan, Tokyo; 29th edn, >989, pp. 424-42s. 11 Y. Yamamoto, Study of the ampemmetric ion-selective electrode sensors, Ph.D. thesis (Agric. Sci.), Kyoto University, 1991, pp. 60-72.