Appl Microbiol Biotechnol (1991) 34:473-477 017575989100009Y

Applied Microbiology Biotechnology © Springer-Verlag 1991

A yeast biosensor for glucose determination Jaroslav Racek Department of Clinical Biochemistry, University Hospital, 305 99 Plzef~, Czechoslovakia Received 18 December 1989/Accepted 23 July 1990

Summary. A y e a s t p o t e n t i o m e t r i c b i o s e n s o r for g l u c o s e d e t e r m i n a t i o n is d e s c r i b e d . A f t e r i n d u c t i o n o f g l y c o lytic e n z y m e s y n t h e s i s a cell s u s p e n s i o n o f t h e y e a s t Hansenula anomala is r e t a i n e d in c a l c i u m a l g i n a t e gel o n the s u r f a c e o f a glass e l e c t r o d e . This b i o s e n s o r gives a N e r n s t i a n r e s p o n s e in g l u c o s e c o n c e n t r a t i o n o f 5 . 1 0 - 4 - 5 • 10 - 3 m o l / 1 with a r e s p o n s e t i m e o f 5 rain a n d a l i f e - t i m e o f at least 2 m o n t h s . M a n n o s e a n d fructose are the only significantly interfering substances. T h e b i o s e n s o r was u s e d f o r m e a s u r e m e n t o f g l u c o s e c o n c e n t r a t i o n in u r i n e with results c o m p a r a b l e to t h o s e obtained by a photometric enzymatic method.

Introduction D e t e r m i n a t i o n o f g l u c o s e in b i o l o g i c a l m a t e r i a l is o f a g r e a t i m p o r t a n c e b o t h in m e d i c i n e a n d the f o o d i n d u s try. E l e c t r o c h e m i c a l m e t h o d s , b a s e d o n g l u c o s e o x i d a s e immobilization on surfaces of special electrodes, are c o m m o n in m a n y l a b o r a t o r i e s ( V a d g a m a 1981). T h e p o s s i b i l i t y o f m e a s u r i n g g l u c o s e c o n c e n t r a t i o n in t u r b i d s a m p l e s s u c h as b l o o d is t h e i r g r e a t e s t a d v a n t a g e . Rec e n t l y s e v e r a l g l u c o s e b i o s e n s o r s with i m m o b i l i z e d w h o l e cells h a v e b e e n d e s c r i b e d . I n c o m p a r i s o n w i t h enzyme biosensors they do not need enzyme isolation, purification and stabilization on the electrode surface a n d t h e i r p r e p a r a t i o n is v e r y s i m p l e a n d c h e a p . G l u cose-metabolizing bacteria (Grobler and Rechnitz 1980; G r o b l e r a n d v a n W y k 1980; K a r u b e et al. 1979; Vais et al. 1985), y e a s t s ( M a s c i n i a n d M e m o l i 1986) o r a m o u l d m y c e l i u m (Vinck6 et al. 1984) with v a r i o u s k i n d s o f d e t e c t i o n h a v e b e e n u s e d for p r e p a r a t i o n o f t h e s e b i o s e n s o r s . I n o u r s t u d y we d e s c r i b e a y e a s t b i o s e n s o r b a s e d o n the a e r o b i c y e a s t Hansenula anomala a n d a glass e l e c t r o d e as t h e m o s t c o m m o n t y p e o f e l e c t r o d e in clinical l a b o r a t o r i e s .

Materials and methods Chemicals. Standard glucose solutions were prepared by dissolving D-glucose in working buffers: imidazole and sodium phos-

phate buffers (0.5 mmol/l), pH 7.4 and 8.0 were tested. All chemicals were purchased from Lachema (Brno, (~SFR) except for imidazole (Serva Feinbiochemica, Heidelberg, FRG) and sodium alginate (Bellco Biotechnology, Vineland, N.J., USA). All chemicals used were of analytical grade. The glucose concentration in urine was measured photometrically after dillution with distilled water (1:100); using Oxochrom Glucose (Lachema) as a reagent kit.

Yeast culture. The aerobic yeast Hansenula anomala, purchased from the Institute of Chemical Technology in Prague, was cultured on Sabouraude's agar and then inoculated into liquid medium. The culture medium used was that of Baudras and Spyridakis (1971) except that L-lactate was replaced by D-glucose (0.3 mol/1) and trace elements were added (Racek and Musil 1987). After incubation at 25°C for 48 h, glucose (0.3 mol/1 in 0.1 mol/l sodium phosphate buffer, pH 6.0) was added (1:4) to the culture and incubation continued for 12 h. During growth the culture was shaken on a linear shaker (50 beats/min). The cells were recultured three times to get the highest induction of glucose-converting enzymes. The yeast cells were harvested by centrifugation (1000 g for 15 rain), washed three times with physiological saline and then resuspended in the same volume of sodium alginate solution (3.2% in physiological saline). This viscous suspension was stored at + 4 ° C. Biosensor preparation and procedures. The cell suspension in sodium alginate solution (ca. 50 ~1) was applied by brush on the surface of the bulb of a glass electrode GA 50N (Research Institute, Meinsberg, GDR) to give a thin compact layer. Then the bulb was immersed into a CaCI2 solution (50 mmol/1 in physiological saline) for 2 h at room temperature and at + 5° C for a further 22 h. During this time a solid calcium alginate gel developed (Ogbonna et al. 1989). Measurements were carried out in a thermostatted vessel with 20 ml imidazole buffer (0.5 mmol/l), pH 7.4, with CaCI~ (50 mmol/l) under constant stirring. The temperature was kept at 25 ° C. The decrease in pH after addition of 0.2 ml glucose solution was observed using a digital pH meter, MV 870 (Pr~icitronic, GDR), and registered by a linear recorder, TZ 4200 (Laboratorni p~istroje, Prague, (~SFR). When biological material was used for glucose determination, the pH change of a second auxiliary electrode with heat-inactivated cells (60°C for 15 min) was subtracted from the result obtained by the working electrode. After the response had reached steady state, the biosensor was immersed into a new portion of working buffer, which was used for the next measurement as soon as the pH value returned to its original level.

474 After measurements the biosensor was stored either in working buffer at + 4 ° C or in the same solution containing glucose (10 mmol/l) at room temperature; this solution was renewed daily.

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Figure 1 shows a calibration graph obtained with the yeast biosensor under the finally recommended conditions. The lower limit of glucose detection is 5.10 -5 mol/l. The biosensor gives a Nernstian type of response in the concentration range 5- 10-4-5.10-3 mol/ 1. Glucose concentrations higher than 2- 10 -2 mol/1 do not lead to a further increase in the biosensor response. Reproducibility defined as a coefficient of variation was 5.7% when a sample with a glucose concentration of 5 mmol/l in the measuring vessel was measured 25 times in a series.

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200 400 U-glucose (rnrnot/tl-phofometric mefhod Fig. 3. Comparison of determination of glucose concentration in urine by a yeast biosensor and by an enzymatic photometric method (n = 35, r = 0.985, y = 11.0 + 1.038x)

The time course of the biosensor response is presented in Fig. 2. The steady state was reached in approximately 5 min; after rinsing the measuring vessel with a new volume of working buffer, 3-5 min were necessary for the pH to return to its original value. The glucose concentration in 35 samples of urine of diabetic patients was estimated with both the yeast biosensor and a routine photometric method (with glucose oxidase, reagent kit Oxochrom Glukosa). The results were compared by linear regression (Fig. 3) and a t test for pair values. No significant differences between the results were observed.

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Two different buffers were tested: sodium phosphate and imidazole buffer, both at pH 7.4 and 8.0. Their concentration increased from 5.10 -4 to 10 -2 mol/l. In

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Fig. 2. Time course o£ the yeast biosensor response to glucose (10 -2, 2.10 -~ and 10-~ tool/l) in imidazol~ buffer (0.~ retool/l), pH ~.4, at 2~°C

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10-3 I0~2 5. I0-2 Buffer concenfrafion (rno[/l) Fig. 4. Dependence of the yeast biosensor response to glucose (]0 mmo]/]) on buffer concentration at 25°C: O - O, phosphate but'fer, pH ?.4; • . . . . • , imidazob buffer, pH ?.4

475 both cases, calcium chloride was added to the end concentration of 50 m m o l / l ; if buffer without Ca z+ ions was used, the alginate gel grew softer and was lost from the electrode surface after several minutes of stirring. There was no difference found between buffers of p H 7.4 and 8.0. Figure 4 shows the dependence between the buffer concentration (pH 7.4) and the biosensor response to glucose (10 m m o l / l ) : with increasing buffering capacity of the working solution the response decreased rapidly in both types of buffers. While phosphate buffer gives turbidity with calcium chloride, imidazole buffer remains clear at all concentrations tested. The precipitation of calcium phosphate leads to a lack of calcium ions and instability of the alginate gel.

Table 1. Interference of some substrates (10 mmol/l) with measur-

ements of glucose concentration (10 mmol/1) by means of the yeast biosensor Substrate

pH

Percentage response to glucose

Glucose Mannose Fructose Maltose Galactose Ribose Xylose Saccharose Lactose Ethanol Acetone Lactate

0.737 0.543 0.501 0.270 0.085 0.039 0.031 0.000 0.000 0.078 0.025 0.000

100.0 73.7 68.0 36.6 11.5 5.3 4.2 0.0 0.0 10.6 3.4 0.0

Influence of temperature The temperature dependence of the biosensor response is shown in Fig. 5. The increasing sensitivity from 20 to 42°C was followed by a rapid fall in sensitivity at temperatures above 45 ° C.

The results are expressed as a decrease in pH and are related to the biosensor response to glucose at the same concentration

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Various m o n o - and disaccharides were tested for possible interference. The yeast biosensor registered the greatest fall in p H in the presence of m a n n o s e and fructose; other saccharides influenced the biosensor to a lesser degree and saccharose or lactose did not interfere with the measurements at all. A small decrease in p H was observed by addition of ethanol and acetone while L-lactate did not cause any biosensor response. The concentration of all c o m p o u n d s tested was 10 mmol/1. The selectivity measurements were m a d e both with the pure substances and in the presence of glucose (10 mmol/1). Both procedures gave similar results; the average values are summarized in Table 1.

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Biosensor age (weeks} Fig. 6. Stability of the yeast glucose biosensor as a response to glucose (10 mmol/1) under two different storage conditions: © --©, biosensor stored at +4 ° C in imidazole buffer (0.5 mmol/ 1), pH 7.4; • . . . . O, biosensor stored at room temperature in the same buffer with glucose (10 retool/l)

Stability of the biosensor

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The biosensor stability was tested under two different conditions. First the biosensor was stored in a refrigerator at + 4 ° C, immersed in working buffer. Second the biosensor was stored at room temperature in working buffer with glucose. Under both conditions the same biosensor life-times were achieved: the response to glucose remained unchanged with small fluctations of 16% for at least 2 months (Fig. 6). The system was in use once a week; each series comprised about 40 measurements o f biological samples or standards.

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Fig. 5. Influence of temperature on the yeast biosensor response to glucose (10 mmol/1) in imidazole buffer (0.5 mmol/1), pH 7.4

Discussion

The principle of microbial glucose biosensors is the glycolytic transformation of glucose except for the Asper-

476

gillus niger-based biosensor, which converts glucose by glucose oxidase in the mycelium (Vinck6 et al. 1984). This glycolytic transformation leads to oxygen consumption and carbon dioxide production. Therefore different electrodes can be used for the detection of glucose transformation by microbial cells. The sensing system of biosensors with Pseudomonas fluorescens (Karube et al. 1979; Vais et al. 1985) is a Clark's oxygen electrode. The biosensors with Streptococcus mutans (Grobler and van Wyk 1980) and a mixture of bacterial cells from dental plaque (Grobler and Rechnitz 1980) are based on a glass electrode whereas Mascini and Memoli (1986) worked on a biosensor with Saccharomyces cerevisiae that registered both oxygen and carbon dioxide. In our study we decided to use the aerobic yeast H. anomala connected to a glass electrode as a glucose biosensor. The purpose of our study was to reduce some interference during biosensor work due to changes in oxygen or carbon dioxide pressure. Thus, a biosensor with pH change as a sensing system was investigated. Aerobic culture of the yeasts is necessary when pH is to be registered in yeast-glucose biosensors: anaerobic metabolism leads largely to ethanol production and only small changes in pH can be observed. The use of diluted buffer has two problems: (a) biological material must be considerably diluted (1:100) with working buffer in order not to affect its p H and buffering capacity (Nilsson et al. 1973; Matsumoto et al. 1979; Vadgama 1981); (b) when the p H of a sample differs from that of the working buffer, the "blank" value obtained with an electrode with inactivated yeast cells must be substracted (Kiihn et al. 1987). We chose 25°C as a working temperature; this temperature is usual for yeast cell culture and temperatures above 37°C are said to be sometimes followed with inactivation of enzymes in the cells (Karube et al. 1979). The yeast biosensor sensitivity corresponds with that of other glucose microbial biosensors (Grobler and Rechnitz 1980; Karube et al. 1979; Mascini and Memoli 1986). The sensitivity is suitable for measurement of glucose concentrations in the urine of diabetic patients or in food, but insufficient for blood samples. The response time of 5 min is a result of the thin gel layer on the surface of the glass electrode, which does not represent a barrier to substrate diffusion. This is much quicker than in any other glucose cell biosensors where the cells are held on the electrode surface by a semipermeable membrane and a steady state is reached in 10 min (Karube et al. 1979), 7-13 min (Mascini and Memoli 1986) or even in 20-40 min (Grobler and Rechnitz 1980). The reproducibility of 5.7% is acceptable for practical use and is considered to be identical to routine photometric methods. Interference of other compounds represents one of the greatest problems in the practical use of microbial biosensors. No glucose biosensor described is specific with respect to other saccharides. The biosensor with P. fluorescens showed a response not only to glucose, but also to galactose, fructose and saccharose (Karube et al. 1979). The yeast biosensor with S. cerevisiae gave a sig-

nificant response to saccharose, glucose, fructose and maltose (Mascini and Memoli 1986). Due to the metabolic activity of Streptococcus mutans, the response to glucose, mannose and fructose was the same whereas galactose and xylulose interfered only in high concentrations (Grobler and van Wyk 1980). The dental-plaque-based biosensor was even less specific because of its ability to metabolize galactose (Grobler and Rechnitz 1980). Although fructose and mannose and to a lesser extent some other saccharides influence our yeast biosensor (Table 1), it seems to be more specific than the biosensors mentioned above. Interference of ethanol and keto-bodies is negligible. Because cells of the aerobic yeast H. anomala are able to metabolize L-lactate (Kulys and Kadziauski6n6 1980; Racek and Musil 1987; Vinck6 et al. 1985), this compound must be considered a potentially interfering substance. No response to lactate was registered by our type of biosensor. Practically all glucose biosensors are stored refrigerated, immersed in an appropriate buffer; their lifetime is said to be 4-5 days (Grobler and Rechnitz 1980; Grobler and van Wyk 1980), or even 1 month (Karube et al. 1979; Mascini and Memoli 1986). The purpose of this procedure is to minimize microbial metabolism and can be used also for our yeast biosensor. Another approach is to store the biosensor at room temperature in a buffer with added glucose: yeast metabolism is stimulated and the cells can grow and multiply. This method of biosensor activity preservation appeared to be useful and we observed stability of the biosensor response for at least 2 months. The yeast biosensor with H. anomala can be used for glucose determination in various biological fluids, especially in urine. Its main advantage is simple operation and low cost of measurements in comparison with a conventional photometric enzyme method. The biosensor has a long life-time and can be easily prepared.

References Baudras A, Spyridakis A (1971) Etude de la L(+)lactate: cytochrome c oxydor+ductase (cytochrome b2) de la levure Hansenula anomala. Biochimie 53:942-955 Grobler SR, Rechnitz GA (1980) Determination of D(+ )glucose, D(+)mannose, D(+)galactose or D( -- )fructose in a mixture of hexoses and pentoses by use of dental plaque coupled with a glass electrode. Talanta 27:283-285 Grobler SR, Wyk CW van (1980) Potentiometric determination of D(+ )glucose, D(+)mannose or l~(--)fructose in a mixture of hexoses and pentoses, by using Streptococcus mutans fermentation. Talanta 27:602-604 Karube I, Mitsuda S, Suzuki S (1979) Glucose sensor using immobilized whole cells of Pseudomonas fluorescens. Eur J Appl Microbiol Biotechnol 7:343-350 Kiihn M, Hamann H, B6ttcher N, Scheller F, Uffrecht E, Brunner J (1987) Analytical systems with potentiometric enzyme electrodes. Stud Biophys 119:171-174 Kulys J, Kadzinauski6n~ K (1980) Yeast BOD sensor. Biotechnol Bioeng 22:221-226 Mascini M, Memoli A (1986) Comparison of microbial sensors based on amperometric and potentiometric electrodes. Anal Chim Acta 182:113-122

477 Matsumoto K, Seijo H, Watanabe T, Karube I, Satoh I, Suzuki S (1979) Immobilized whole cell-based flow-type sensor for cephalosporins. Anal Chim Acta 105:429-432 Nilsson H, Akerlund A-Ch, Mosbach K (1973) Determination of glucose, urea and penicillin using enzyme-pH-electrodes. Biochim Biophys Acta 320:529-534 Ogbonna JC, Amano Y, Nakamura K (1989) Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate. J Ferment Bioeng 67:92-96 Racek J, Musil J (1987) Biosensor for lactate determination in biological fluids. I. Construction and properties of the biosensor. Clin Chim Acta 162:129-139

Vadgama P (1981) Enzyme electrodes as practical biosensors. J Med Eng Technol 5:293-298 Vais H, Oancea F, Fagliu AM, Delcea C, Margineanu DG (1985) Amperometric electrode for glucose with immobilized bacteria (Pseudomonasfluorescens). Rev Roum Biochim 22:55-63 Vinck~ BJ, Kaufmann J-M, Devleeschouwer MJ, Patriarche GJ (1984) Nouveau module d'61ectrode enzymatique pour la d~termination du glucose. Analusis 12:141-147 Vinck6 BJ, Devleeschouwer MJ, Patriarche GJ (1985) Electrodes potentiometriques et amperometriques a levures permeabilises: determination du L-lactate. Anal Lett 18 : 593-609

A yeast biosensor for glucose determination.

A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast...
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