a very simple biosensing system for educational...

Chem. Anal. (Warsaw), 51, 977 (2006)

Keywords: Biosensor; Urea; Potentiometry; Teaching

A Very Simple Biosensing System for Educational Purposes

by £ukasz Tymecki*, Beata Rozum and Robert Koncki

University of Warsaw, Department of Chemistry,ul. Pasteura 1, 02-093 Warsaw, Poland

An extremely cheap, complete bioelectrochemical cell for potentiometric determination ofurea has been described. The cell consists of two pH-sensitive metal oxide electrodes: oneof them plays a role of an internal electrode of the biosensor, whereas the second one servesas a pseudoreference electrode. Either enzyme or biological material containing urease hasbeen immobilized physically by entrapment in polymer membrane. For potentiometric mea-surements with such a biocell, a simple and low-cost multimeter is sufficient. The proposedvery simple biosensing system is useful in students� laboratory practice to illustrate severalproperties of biosensors, i.e. biosensitivity, dynamics, determination range, operational andstorage stability, benefits of the applied immobilization methods, etc.

W pracy przedstawiono bardzo prosty do wykonania system potencjometryczny do biode-tekcji mocznika. Biogniwo pomiarowe sk³ada siê z dwóch tlenkowych elektrod pH-metrycz-nych, z których jedna pe³ni funkcje elektrody pseudoreferencyjnej, za� druga elektrodywewnêtrznej bioczujnika mocznikowego. Do uczulenia tej elektrody wykorzystano fizycznemetody immobilizacji unieruchamiaj¹c inkluzyjnie oczyszczony enzym b¹d� tkankowy pre-parat biologiczny zawieraj¹cy ureazê. Stwierdzono, ¿e do pomiarów potencjometrycznychz u¿yciem tak otrzymanego bioogniwa mo¿na zastosowaæ tani miernik elektroniczny. Opi-sany system bioanalityczny mo¿e s³u¿yæ do eksperymentalnej weryfikacji zasady dzia³aniabioczujników oraz okre�lenia wp³ywu ró¿norodnych parametrów i czynników na charakte-rystyki analityczne bioczujników. Znacz¹cymi zaletami zaproponowanego systemu s¹: jegoprostota, znikomy koszt oraz mo¿liwo�æ wykonania pracy eksperymentalnej (wytworzeniesystemu analitycznego oraz badanie jego charakterystyk analitycznych) bez stosowaniazaawansowanego sprzêtu laboratoryjnego oraz kosztownych odczynników.

* Corresponding author. E-mail: [email protected]

978 £. Tymecki, B. Rozum and R. Koncki

Biosensors constitute a significant group of analytical tools important in severalfields of modern analytical and bioanalytical chemistry. Nearly all types of biochemi-cal and biological interactions can be applied in modern analyte recognition schemes.In these recognition schemes may participate diverse species, including small biomole-cules, drug candidates, carbohydrates, membrane receptors, bioactive proteins, enzy-mes, antibodies, nucleic acids, whole bodies of viruses, cells, and microorganisms.Representative example of such analytical applications is commercial success of SPR--based biosensing systems developed by Biacore [1]. A flagship example of success-ful biosensor developments are glucose-sensing devices for diabetes. Over forty yearshave passed since Clark and Lyons proposed glucose enzyme electrodes [2]. Now,more than 25 companies offer analytical systems for diabetes � nearly all of them arebased on glucose biosensors. Forty years of advances and challenges in the field ofglucose biosensors as well as the corresponding current commercial aspects havebeen recently reported by Wang [3] and Newman and Turner [4]. Medical applica-tions of biosensors overshadow other important applications areas. Pharmaceuticalresearch industry is constantly driving a need for new rapid assay biosensors to speedthe progress of drug design. Security problems drive a need for new rapid detectionbiosensors against bio-warfare agents for military and civil defense applications.Biosensors help to monitor food safety and detect environmental pollution. In thecommercial report by Fuji-Keizai [5] it has been estimated that the worldwide marketin biosensors at the end 2003 was about $7.3 billion and is anticipated to grow up toabout $10.8 billion in 2007 at a growth rate of about 10.4%.

Owing to such great role played by biosensors in modern analytical chemistry itis important to provide appropriate academic courses including theoretical principlesas well as laboratory practice concerning biosensors. Obviously, experiments carriedout by students allow them to better understand the idea of construction and workingprinciples of these attractive analytical tools. Unfortunately, bioreceptors are usuallyexpensive or/and unstable. Moreover, the use of some transducer systems needs sophi-sticated instrumentation and qualified personnel. Finally, some methods of biosensorpreparation are complex and time-consuming. Taking into account all these facts, it israther difficult to bring the concept of biosensors to the students� labs or high schoolclasses. In this paper, an example of a simple, easy-to-prepare, reproducible, andalmost costless biosensing system for educational purposes has been described.

The concept of a biosensing system

The main goal of this study was to develop a biosensor, or �- more precisely �a complete biosensing analytical system, easily understandable by students, easy-to--adapt to their experimental settings, and clearly demonstrating general biosensorproperties. The second requirement implicates the need of experimental simplicity of

979A very simple biosensing system for educational purposes

the system and reduction of outlays. In practice it means that the use of reagents andinstrumentation should be mostly reduced. Moreover, the whole experiment, inclu-ding preparations and measurements should not be complicated, tedious, and time--consuming.

Taking into account all these demands, we have developed very simple potentio-metric biosensors for urea based on pH-metric metal-metal oxide electrodes. In thecourse of enzymatic reaction catalyzed by urease, urea is hydrolyzed to form alkalineproducts according to the following reaction:

CO(NH2)2 + 3H2O ® 2NH4+ + HCO3

� + OH�

The resulting increase of pH in microenvironment of the internal electrode causesa decrease of its potential. Clearly, potential of a biosensor is a function of urea con-centration.

Reference electrode is indispensable for potentiometric measurements. To com-plete the developed electrochemical cell, an identical pH-electrode without biocatalyticlayer has been applied as a reference electrode. Under specified conditions (i.e. con-stant pH of the working buffer solution), such pH-sensor acts as a pseudoreferenceelectrode as the pH shift accompanying enzymatic reaction takes place only withina biocatalytic layer covering the internal electrode of the urea biosensor. Constant pHin the bulk of the working buffer solution determines constant potential of pseudo-reference electrode. Thus, for elementary student experiment any commercial refe-rence electrode is unnecessary. A detailed description of the preparation of electrodesis given in next paragraph.

Owing to the need of noise-free measurements in precise potentiometric experi-ments, sophisticated high-impedance potentiometers are recommended. However,the bioelectrochemical cell reported in this work consists of two low-resistive metal--metal oxide electrodes and therefore the use of a simple multimeter is sufficient.This way, the measurements in student exercise can be performed using extremelycheap instrumentation (the cost of simple multimeter does not exceed 10$). The schemeand the photo of the measurement system used for basic student�s experiment aregiven in Figure 1.


Basic student experiment

Both the internal sensor of a biosensor and a reference electrode are based onantimony oxide electrodes prepared by students in the same way. Firstly, heat-shrin-kable tubings were put on the stainless steel screws (4 mm in diameter, 35 mm inlength) to determine the working surface of the electrode. Then, the screws wereimmersed into the saturated antimonyl potassium tartrate (emetic) solution and elec-trolysis was carried out for a few minutes to cover the screws with black metallicantimony. Ordinary 9-Volt battery was used as the power source. As an anode, the

�

Figure 1. The scheme and the photo of the experimental setup. Photo-inset: a screw (a), a pH-electrode (b),a biosensor (c)


identical screw was applied. Electrolysis potential and time were not strictly con-trolled. After the electrolysis, the screws with deposited antimony were washed withdistilled water and kept in air at the room temperature for about one quarter. Duringthis time, the surface of antimony was spontaneously oxidised. The screws serving asreference electrodes were immersed for a moment in 1% (w/w) cellulose acetatesolution in acetone, and dried for half a minute. For fabrication of a biosensor,a similar procedure was carried out, yet cellulose acetate solution contained additio-nally the enzyme (10 mg mL�1, urease powder 60 U mg�1) and the screws were twicedipped into this suspension. Thus, to prepare a biosensor, a simple physical methodof enzyme immobilization was applied. Urease was trapped into a cellulose acetatematrix. All the electrodes were conditioned for 15 min in phosphate buffer solution toremove an excess of weakly immobilized biological material. This easy-to-preparebuffer solution (without using pH-meter; obtained by dissolution of 1 mmol ofNa

2HPO

4, 10 mmol of NaH

2PO

4, and 100 mmol of NaCl in 1 L of distilled water) was

used also for the measurements. After conditioning, the electrodes were ready formeasurements. They were mounted into Lego� blocks to construct the measure-ment cell and connected to the multimeter. This very simple and complete measure-ment setup is shown in Figure 1. Calibration of the biocell was performed by stepwiseaddition of the urea standard to the working buffer. The changes of the electromotoricforce generated by the biocell were measured as an analytical signal. The results oftypical calibration and the corresponding calibration plot are shown in Figure 2a.

The arrangement of the biosensing system described above can be modified depen-ding on the availability of the components. First of all, metal electrodes can be repla-ces with other material. For example, we have tested ordinary two-core copper cableas the biocell body and similar results have been obtained. In such case, electrolessdeposition of metallic antimony from strongly acidic solution of emetic is possible.Unfortunately, pH-sensitivity of the resulting metal oxide was significantly worse.Noteworthy, many other easily available/preparable pH-sensitive materials can beapplied at this stage of experiment, like chemically or electrochemically depositedfilms of polypyrrole, polyaniline, etc.


��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

E�

��PPRO�/

±

�

��PPRO�/

±��

��PPRO�/

±��

��PPRO�/

±��

��PPRO�/

±�

D�

��PPRO�/

±��

��PPRO�

/

±�

��PPRO�/

±��PPRO�/

±��

��PPRO�/

±��

7LPH��PLQ�

��

(0)��P9�

'(

0

)

�

�

�

P

9

�

S85($�

S85($�

'(

0

)

�

�

�

P

9

�

Figure 2. Calibration of biocells and the corresponding calibration plots (insets): (a) urease-based biosen-sor, and (b) soybean-based biosensor

As matrix materials for enzyme immobilization several polymers, earlier reportedas useful for such purposes [6], can be used. Membranes deposited from acetonesolutions of cellulose derivatives (ethylcellulose, triacetate cellulose and trinitratecellulose) were tested and the obtained films exhibited similar properties. The mem-branes deposited from THF solutions of polyvinylchloride and its derivatives (likechlorinated and carboxylated PVC) were more robust, however, the required condi-tioning time, response time, and return time were significantly longer. Such observa-tions have confirmed the previous findings [6]. It is worth noticing that at this stageof basic experiment it is possible to prepare biosensing membranes with different


immobilization matrices and of different thicknesses as well as various enzyme acti-vities. Further measurements with these bioelectrodes might be a good experimentalexample how the analytical parameters of biosensors (mainly sensitivity, dynamicsand stability) depend on the biosensing membrane properties.

The most expensive component of the proposed system seems to be urease. How-ever, suspensions of the enzyme in acetone and THF are relatively stable. Therefore,once prepared mixture for biosensing layer deposition can be used by consecutivegroups of students for a long time. The reported exercise was repeated for severaltimes during three months using the same suspension, and the prepared biosensorsexhibited similar properties. Only 2 mL of the suspension (equivalent to 20 mg of theenzyme) was sufficient for all groups (over 100 biosensors were prepared). More-over, for the preparation of a biocatalytic layer, less active enzyme can be used. In theextreme case, a biological material containing the enzyme can be applied instead ofa purified protein. Such an approach allows one to prepare a so-called microbial/tissue biosensors. In the course of the proposed exercise, urease powder can be re-placed with finely grounded soybean. The amount of meal obtained from fresh soy-bean was 200 mg mL�1 in the suspension in 1% TAC acetone solution. The results oftypical calibration and the corresponding calibration plot of such tissue-basedbioelectrochemical cell are shown in Figure 2b. Obviously, such biosensing systemexhibits slightly worse analytical parameters (lower sensitivity and longer responsetime) than the enzyme-based bio-device (Fig. 2a), but still satisfactory for educa-tional purposes. Moreover, experimental comparison of tissue- and enzyme-basedbiosensors, especially their dynamics, is interesting from educational point of view.

Extension of basic experiment

Basic students� exercise described in the previous section can be easily extended.Taking into account that only the properties of the biosensing layer determine analy-tical parameters of the resulting biosensors, it is reasonable to test several methods ofbiosensor layer preparation. Other possible experiments which do not require anyadditional expenses include the studies on the influence of experimental conditionson the biosensor performance. It has been experimentally confirmed that the analyti-cal response of the developed biosensors can be satisfactorily described by the respec-tive theories. The effect of pH and working buffer solution capacity on the shape ofthe calibration plots of the biosensors are consistent with theoretical predictions basedon the models of pH-based enzyme electrodes [7]. These effects can be experimen-tally observed and then easily explained by students considering protolytic equilibriawithin a biosensing layer.

In the extended students� exercise, when additional instrumentation is available,students can perform a more detailed study on the properties of electrodes. Antimony


electrodes, playing a double role in the prepared biocells, can be analytically charac-terized in the course of pH-metric titration applying a standard reference electrodeand a calibrated pH-glass electrode. As shown in Figure 3a, the results of such titra-tions allowed one to evaluate analytical parameters of the sensors (i.e. sensitivity,linearity, time of potentiometric pH-response). If a standard reference electrode isavailable, students also may check the behaviour of the prepared biosensors andpseudoreference electrodes during calibrations on urea. As shown in Figure 3b, theycan observe that the potential of biosensors is a function of urea concentration, whereasthe potential of pseudoreference electrodes is constant during calibration. This expe-riment confirms that pH-electrode without the enzyme layer can be used as a pseudo-reference electrode and that the changes of biosensor�s potential are due to pH changesand occur only in the enzyme layer immobilized at the electrode surface. If pseudo-reference electrode exhibits a response to urea, this means that the enzyme has beeninsufficiently immobilized and biocatalytic reaction takes place also in the bulk solu-tion and not only in the biosensing membrane.

��

�

��

��

�

��

��

��

��

��

��

�

��

��

�

��

�

��

��

�

��

��

��

��

��

��

��

��

��

��

��

��

��

�

��

�

��

��

��

��

�

(

0

)

�

�

�

P

9

�

7LPH�

��PPRO�/

±��

��PPRO�/

±��

��PPRO�/

±��

��PPRO�/

±��

��PPRO�/

±��

��PLQ�

E�

D�

S85($�

D�

E�

(

0

)

�

�

�

P

9

�

S+�

(

0

)

�

�

�

P

9

�

'(

0

)

�

�

�

P

9

�

Figure 3. pH-sensitivity (left) and urea-sensitivity of metal oxide electrodes with (a) and without (b)the enzyme layer (right). The corresponding calibration plots are shown in the insets

The proposed measurements help a lot to understand the results of basic students�experiment (biocell operation). If they are performed using a multichannel data acqui-sition system, reproducibility of pH-electrodes and biosensors prepared by studentscan be evaluated in a single experiment (Fig. 3).


CONCLUSIONS

Simple and almost costless experiments reported in this work are helpful in betterunderstanding of biosensing principles that are utilized also in more advanced analy-tical tools. For example, nearly the same (bio)sensing and detection scheme is real-ized in planar microbiodevices for determination of metabolite in physiological flu-ids like urine and blood [8]. Similarly manufactured thick-film voltamperometricdevices are commercialized as strip glucose biosensors for diabetes [4]. Further inves-tigations on other kinds of sensors and biosensors useful for educational needs are inprogress.

Acknowledgements

The participation of the IV-year students (University of Warsaw, Department of Chemistry) in theexperimental work is kindly acknowledged. This contribution was accomplished in the frame of the Angus-MacGyver Project and is dedicated to the Deans of our Department.

REFERENCES

1. www.biacore.com2. Clark L. and Lyons C., Ann. NY Acad. Sci., 29, 102, 19623. Wang J., Electroanalysis, 13, 983, 20014. Newman J.D. and Turner A.P.F., Biosens. Bioelectron., 20, 2435, 20055. Biosensor Market, R&D and Commercial Implication, Fuji-Keizai Report, April 20046. Koncki R., Leszczyñski P., Hulanicki A. and G³¹b S., Anal. Chim. Acta, 257, 67, 19927. G³¹b S., Koncki R. and Hulanicki A., Analyst, 117, 1675, 19928. Tymecki £., Zwierkowska E. and Koncki R., Anal. Chim. Acta, 538, 251, 2005

Received May 2006Accepted September 2006

a very simple biosensing system for educational...

Documents