Optosensing of d -glucose with an immobilized glucose oxidase minireactor and an oxygen room-temperature phosphorescence transducer

439

Analytica Chimica Acta, 283 (1993) 439-446 Elsevier Science Publishers B.V., Amsterdam

Optosensing of D-glucose with an immobilized glucose oxidase minireactor and an oxygen room-temperature phosphorescence transducer M.J. Valencia-Gonzalez, Y.M. Liu, M.E. Diaz-Garcia and A. Sanz-Medel Departmentof Physical and Analytical Chemistv, Faculty of Chemhy, University of Oviedo, Au. Jtin Ckzveria 8, 33006 oviedo (Spain) (Received 7th October 1992; revised manuscript received 20th January 1993)

Glucose oxidase was immobilized covalently on a nylon membrane and the oxygen consumption was followed via the changes in the room-temperature phosphorescence. (RTP) of an oxygen-sensitive metal chelate whose RTP is quenched dynamically by molecular oxygen. The metal chelate was immobilized on an anion-exchange resin and packed into a conventional flow-through cell. As result of the glucose oxidation in the presence of the enxyme, a certain amount of oxygen was consumed, which in turn was indicated by the RTP of the chelate. Measurements were made in flowing air-saturated solutions containing 0.1 M acetate buffer (PH 5.9). The system is linear for 0.5-2.5 mM glucose with a relative standard deviation of 1.6% at 2 mM (ten measurements). The detection limit is 8 x lo-’ M and the application of the system to glucose determhration in serum and beverages is demonstrated. &NW&:

Biosensors; Enxymatic methods; Flow injection; Phosphorimetry; Glucose; Gxygen

Glucose is among the most frequently determined sugars, especially in clinical samples for the diagnosis of disorders of metabolism [ll and in the food and biotechnology industries for process monitoring. Conventional chemical methods for determining glucose are based on its reducing properties and so are unselective. Therefore, schemes based on liquid chromatography have been proposed [2] to enhance the selectivity. The use of enzymes and sensors offers a less expensive approach based on enzyme selectivity (and occasional specificity) Correspondence to: A. Sanx-Medel, Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Gviedo, Av. Juli&r Claverfa 8, 33006 Gviedo (Spain). 0003-2670/93/$06.00

and capability of self-regeneration via the catalytic cycle. There has been much interest in the development of enzyme electrodes for ducose in which oxygen consumption, hydrogen peroxide production or gluconic acid production, resulting from the glucose oxidase reaction, are detected electrochemically by using an oxygen, H,O, or pH electrode, respectively. Of particular practical interest are amperometric enzyme electrodes employing a synthetic mediator (ferrocenes, phenoxazine, quinones, etc.) [3-61, which acts as a shuttle for electrons between the enzyme active site (reaction centre) and the electrode which renders such sensors ahnost inert towards variations in naturally available (dissolved) oxygen back-

0 1993 - Elsevier Science Publishers B.V. All rights reserved

440

ground. General advantages of optical over electrochemical transduction have been discussed in several papers [7-91, but so far optical glucose sensors are impractical and more work is needed in order to be able to apply them to real analyses. Schultz et al. [lo] proposed one of the first optical sensing principles for glucose in their “glucose affinity sensor” based on the competition between sample glucose and fluorescein-labelled dextran for binding sites at surface-immobilized concanavalin A. Uwira et al. [ll] described a fibre-optic glucose biosensor based on the measurement of enzymatic oxygen consumption via the changes in the fluorescence of an oxygen-sensitive dye. An important drawback of the device is that the fluorescence indicator (pyrenebutyric acid) has an excitation wavelength in the UV region, not compatible with the transmission characteristics of economic glass or plastic optical fibres. Moreover, slow leaching out of the indicator was observed. Another type of optical glucose sensor that uses coloured or fluorescent dyes but this time as pH indicators has also been proposed [12,13]. More recently, a glucose biosensor based on the intrinsic green fluorescence of glucose oxidase (GOx), which changes during interaction with glucose, has been developed [14]. Although this sensor is direct (in that it does not need a transducer element), the fairly low intensity of the enzyme fluorescence and the small glucose concentration range in which fluorescence intensity changes occurred severely limit its practical use. Recently, Papkovskii and co-workers [15-171 described some metal phorphyrin chelates for 0, sensing based on room-temperature phosphorescence (RTP). A sensitive RTP flow-injection method for glucose was developed based on the use of a column of immobilized GQx and the room-temperature phosphorescent indicators Pt2+-coproporphyrin III and Pd2+-coproporphyrin I as internal molecular oxygen sensors [163. The method allowed the determination of glucose in biological fluids with low protein contents (0.1%). For samples with high protein contents (e.g., serum) the composition of the elution buffer had to be modified in order to decrease the high background luminescence resulting from interac-

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Chim.Acta 283 (1993) 439-446

tion of the oxygen sensor with the sample proteins. Following a similar principle, this paper describes a sensitive flow-injection method for the determination of glucose in food and clinical samples using an immobilized glucose oxidase minireactor. Oxygen consumption during the enzymatic reaction is followed via the changes in the RTP of an oxygen-sensitive metal chelate immobilized on an anion-exchange resin packed into a conventional flow-through cell. The method is simple, consumes only inexpensive reagents (air-equilibrated acetate buffer) and requires no sample preparation for real beverage or serum samples. The use of other enzymes or a combination of two or more enzymes which require oxygen as co-substrate will possibly extend the application of this RTP transducer to other analytes such as ethanol, cholesterol and lactate 118-211, already determined by alternative electrical and/or optical transduction.

EXPERIMENTAL

Chemicals and solutions Glucose oxidase (EC 1.1.3.4, Type II, from AspergiUus niger ) with a specific activity of 25 000 U g-’ was obtained from Fluka. Lysine (97%, w/w), and glutaraldehyde (50%, w/w) aqueous solutions and dimethyl sulphate were obtained from Fluka. a-D-Glucose was purchased from Aldrich-Chemie and 2-W-morpholino)ethanesulphonic acid (MES) and bis(2-hydroxyethylhrninotris(hydroximethylhnethane (Bis-Tris) from Sigma. All other reagents used were of analytical-reagent grade. A glucose VIS-UV-enzymatic kit was obtained from Sigma. All aqueous solutions were prepared in 0.1 M sodium acetate-acetic acid buffer (pH 5.91, using water obtained from a Milli-Q system (Millipore). Glucose solutions were allowed to mutarotate overnight before use. Aluminium-7-iodoquinolin-8-ol-5-sulphonic acid chelate solution This solution was prepared by mixing 0.3 ml of a 1000 pg ml-’ AI stock solution and 35 ml of

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3 x 10e3 M ferron (7-iod~uinolin-8-ol-5-sulphonic acid) in a 100~ml volumetric flask. The solution was diluted to volume with 1 M acetate buffer (pH 5.5). This “chelate solution” did not deteriorate (stable RTP signals) for at least 3 months. Optosensing assembly In a single-line flow-injection system, a Hellma Model 176.52 flow-through cell of volume 25 ~1 was packed with the anion-exchange resin and placed in the conventional sample compartment of the detector (see Fig. 1). An Gmnifrt 1106 rotary valve was used for sample introduction. PTFE tubing (0.5 mm i.d.1 and fittings were used for connecting the flow-through cell, the rotary valve, the enzyme minicolumn and the carrier solution reservoir. All the connections were sealed carefully with high-vacuum silicone grease (BDH) to avoid diffusion of oxygen into the system. A Gilson Minipuls-2 peristaltic pump was used to generate the flow stream. All RTP measurements were made at 600 run (excitation at 390 nm) with a Perkin-Elmer LS5 spectrofluorimeter which employs a xenon-pulsed (10-s half-width 50 Hz) excitation source and is equipped with a Perk&Elmer Model 3600 data station. The delay time used was typically 0.04 ms

for optimization of system variables and 0.05 ms for real sample analyses. The gate time used throughout was 2 ms. The instrument excitation and emission slits were set at 10 and 20 nm, respectively, throughout. Steady-state phosphorescent signals were recorded with a Perk&Elmer Model 560 recorder. Z’reparation of the oxygen transducer For immobilization of the oxygen “indicator”, the Al-ferron solution was pumped through the sensing flow cell containing a basic anion-exchange resin. It was found that after passing the carrier solution for 5-6 h the variation in the RTP intensity observed was less than 4-5%. In any case, it was checked that sensor phase “renewal” was accomplished just by injection of 2 ml of 6 M HCl in order to release “old” Al-ferron chelate from the resin. As detailed above, new Al-ferron solution can be then pumped in order to obtain a new active surface for oxygen sensing. Details of this RTP sensing phase production are the subject of a patent application [22]. Zmmobilization of glucose oxidase on nylon Covalent binding of the enzyme to the inert matrix involved the preparation of the nylon and

SAMPLE CARRIER

-

0.1M HAclNaAcpH 5.9

I

I ENZYME MINICOLUMN

PERJSTALTICPUMP

I

I

I

I

UI DETECTOR

OXYOENSENSOR

Fig. 1. Schematic diagram of the flow system.

G

1 WA5TE

442

reaction with glutaraldehyde as described elsewhere [23]. Coupling was carried out at room temperature for 2 h, after which the membranes were stored in a refrigerator at 4°C overnight. Finally, the excess of enzyme was washed out with “phosphate buffer” followed by 0.1 M NaCl solution in phosphate buffer and then pure phosphate buffer again. When not in use, the enzyme membranes were stored at 4°C in 0.1 M phosphate buffer containing 0.1% sodium azide as bactericide. The enzyme-membrane so obtained was mounted around an open Tygon tube (5 mm i.d.) and inserted in the flow-injection manifold depicted in Fig. 1. The activity after binding of the enzyme was determined by measuring the enzyme activity in the solutions before and after immobilization. General procedures for glucose determination Calibration. Glucose solutions (consisting of carrier solution plus a known volume of the glucose standard solution) were injected through the valve (sampling volume 0.2 ml) into the carrier stream. The flow-rate of the carrier was set at 0.40 ml min-‘. Typical flow-injection RTP signals were measured and peak height was plotted against molar concentration of glucose injected. Serum samples. The serum samples were diluted 1 + 9 with the 0.1 M acetate buffer (pH 5.9). The glucose content was evaluated from calibration graphs obtained using aqueous glucose standards. Comparison was made with results obtained using the Beckman electrode method, routinely used in the Hospital Central de Asturias (from where the samples were obtained). Fruit juice and lemonade samples. Samples of fruit juices with low levels of insoluble ingredients (e.g., fruit pulp) were diluted 200-fold with the acetate buffer solution and injected into the flow system without further treatment. Highly carbonated samples, such as Lemon-Kas, were shaken overnight to decrease the amount of dissolved carbon dioxide because an excess of carbon dioxide disturbs the flow system by generating bubbles. These samples were diluted 500-fold with the buffer solution.

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The glucose content in all fruit juices was evaluated from calibration graphs obtained using aqueous glucose standards. The glucose content in lemonades, however, was evaluated using the standard addition technique because the sample matrix interfered in the detection scheme.

RESULTS AND DISCUSSION

Charactektics of the RTP oxygen transducer The complex formed by ferron and aluminium immobilized on a strong anion-exchange resin exhibits solid-surface RTP (SS-RTP). The corresponding spectra showed an emission maximum at 600 nm with an excitation maximum at 390 nm. Solid-state membranes of this sensor phase, based on silicone, were also developed to construct oxygen-sensitive membranes. The characterization of such a transducer was performed by monitoring both the RTP intensity and/or lifetime measurements during some oxygenation-deoxygenation cycles in aqueous and in gaseous phases [22]. Immobilization of this phosphor on the solid support seems partially to protect the triplet from non-radiactive collisional deactivation by quenchers, even in an aqueous flow. The sensor is completely reversible to oxygen both in gaseous mixtures and aqueous samples and its response to oxygen was linear up to 20% 0, (v/v) in an argon gas stream. Under the optimum experimental conditions typical response times for full signal change were ca. 20 s for gaseous mixtures and ca. 1 min for solution samples. The sensor exhibited no hysteresis and showed high photochemical stability. More information on the analytical performance of this RTP-0, sensing phase is the subject of a patent application [22]. Optimization of experimental conditions The pH and the ionic strength of the solutions used are important parameters in the continuous-flow system described. Experiments were carried out using sodium acetate buffer to establish the optimum pH for the glucose determination. Figure 2 shows the pH profile for the injection of 2 mM glucose. An RTP signal plateau was found at pH 4-5. Commercially available GGx usually

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I

01” 3

I 3.5

4.5

4

5.5

6

6.5

7

Fig. 2. Influence of pH on the RTP signals.

contains other enzymes as impurities, at pH < 5 GGx activity is reduced but other enzymatic reactions could also take place simultaneously. Data are given in Table 1 for two common sugars. Consequently, all the analyses were done at pH 6.0 f 0.1, where GGx activity is high and the oxygen RTP sensor performance is optimum [24]. The effect of the buffer composition is remarkable. MES buffer at pH 5-6 inhibited the enzyme activity gradually with continuous use. Phosphate buffer (1 M) interfered with the oxygen sensor as a continuous flow of phosphate at such a high concentration complexed progresively with aluminium. The behaviours of Bis-Tris and acetate buffer were similar. Therefore, a simple carrier solution of acetate buffer was used throughout.

TABLE 1 Performance of the enzyme minireactor at different pH values Sugar

PH

Glucose a/sugar mole ratio

Ib

Galactose

4.5

1:lO 1:lOO 1:lO 1:lOO 1:l 1:s 1:l 1:lO

108 135 102 112 110 112 100 103

6.0 Fructose

4.5 6.0

a 2 mM glucose. b I = relative RTP intensity.

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On increasing the acetate buffer capacity a double effect was observed. Firstly, higher buffer concentrations (e.g., 1 M sodium acetate) inhibited enzyme activity (to even less than 70% of that achieved with low buffer concentrations). On the other hand, the oxygen RTP signals increased with saline content. This could be explained by taking into account that a higher ionic content could result in a more “rigid” solid support [251 of the oxygen sensor and hence to a more rigid environment that could prevent non-radiational RTP deactivations. A 0.1 M sodium acetate concentration in the acetate buffer was used for remainder of the study. The RTP signals decreased steadily with increasing flow-rate of the carrier solution (from 0.20 to 1.00 ml min-‘1, as would expected owing to the decreased reaction time. A compromise value of 0.40 ml min- 1 was selected as good sensitivity and a relatively short response time were obtained. To investigate the effect of enzymatically generated H,O, on the detector signals, the GOx minicolumn was removed from the FIA manifold. Then increasing concentrations of H,O, (from 0.5 to 50 mM) were injected into the system and the fluorescence [A(ex) = 390 nm, h(em) = 498 nm] of the immobiliied Al-ferron was recorded. No signal changes were observed and only after continuous pumping of a 50 mM H,O, solution for 15 min did the fluorescence intensity decrease by 6% of its original value. Exactly the same effect was observed for the RTP signals. As these high H,O, concentrations are not normal in practical assays, it seems that H,O, will not pose an interference problem for the oxygen detector itself. However, it has been reported 1261that the enzymatically generated H20, inhibits the immobilized GGx activity and so catalase (or peroxidase) is sometimes co-immobilized with GOx to circumvent this problem. The stability of the enzyme column was investigated by comparing the RTP responses observed for 30 repetitive injections of a 2 mM glucose solution. The enzyme was observed to retain only lo-20% of its original reactivity after 25 injections. This inhibition was due to the reversible momentary accumulation of H,O, and

444

M.J. Vaknciu-Godkz

after a 2-3-min flow of the buffer carrier through the membrane the enzyme activity reached its original value. It is worth mentioning that the use of a co-immobilized enzyme (e.g., catalase or peroxidase) in the present system to avoid the H,O, effect could reduce the sensitivity obtainable because oxygen is produced in the catalase reaction, whereas oxygen is consumed in the measured analytical reaction (GOx reaction). Regarding the background of RTP signals (dissolved oxygen level), a constant background level was found when air-equilibrated carrier and/or real samples passed over the RTP sensor. This background level was identical with that observed when the sample was split into two fractions, one passing through the enzyme column and the other avoiding the column and feeding the 0, detector directly. Thus, a constant oxygen supply was always observed with this procedure, and so the analytical signals were obtained by subtracting manually the background close to the value of the RTP total signal measured by the instrument.

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centrations. The calibration graph was linear in the glucose concentration range 0.5-2.5 mM, with a detection limit (estimated on the basis of the 3 times the standard deviation of the blank) of 8 X 10m5 M. The reproducibility of the proposed enzymatic glucose assay system was tested by comparing the responses of ten repetitive injections of a 2 mM glucose solution. The relative standard deviation was 1.6%. The stability of the enzyme minicolumn was also investigated over a 4-month period. The reactor retained 100% of its original activity after 2 months and decrease only to 90% after 4 months. Interference study and analytical application The interferences of fructose, saccharose, lactose and galactose were studied as GOx could be contaminated by other enzyme traces producing interferences in 0, consumption. Different concentration ratios of glucose to these sugars and their effects on the RTP response to glucose at two different pHs were studied. Only galactose gave positive deviations (ca 12%) for a glucose : galactose ratio of 1: 100. This galactose interference was due to galactosidase impurities in the GOx enzyme.

Analytical features Figure 3 shows peak and a typical calibration graph for injections of glucose at different conI(RTP)

25 -

20 15 !

0

3.5

0.5

G~“COS~CONC&lTRA?lOfV

4

(n-h)

Y= -0.46+11.9X

Fig. 3. Calibration graphs for the proposed sensor and response profile [using a 0.2~ml sample loop and 0.1 M acetate buffer (pH 5.911.

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M.J. Valencia-Gonzdez et al. /Anal. Chim. Acta 283 (1993) 439-446 TABLE 2 Analytical applications by direct calibration Sample

Serum pool I Serum pool II Serum pool III Apple soft drink Lemon soft drink d

Glucose concentration (mM) This sensor a

Alternative method

4.85f 5.57* 6.53+ 204.0 f 390.0 f 282.5 f

4.9OkO.12 b 5.64*0.04 b 6.36f0.12 b 210.0 k4.6 ’ 283.4 k9.2 =

0.12 0.02 0.13 0.2 13.3 3.5 e

a Mean f SD. (n = 3). b Beckman oxygen electrode; mean f S.D. (n = 3). ’ Spectrophotometric determination (hexokinase at 340 nm); mean*S.D. (n = 3). d The sample was stirred to remove COz. e Standard addition.

Sulphite or other reducing agents can lead to positive deviatioris of signals because it might react with oxygen (resulting in an enhanced RTP signal) and their possible presence in samples should therefore be taken into account. The effect of possible metals present in biological fluids is not serious using this sensing phase [24] and, in any case, it proved to be negligible in the diluted samples analysed. In order to assess the validity of the system, several serum samples and beverages were analysed for their glucose content following the general procedure detailed under Experimental. The results obtained compared favourably with those obtained by alternative assays, as shown in Table 2. The alternative techniques used for comparison were the Beckman oxygen electrode and W method using the HK procedure (Sigma kit). Measurements were made according to the manufacturer’s instructions. Hence, it was demonstrated that the proposed RTP optosensing method can be used successfully for the determination of glucose in important clinical and food samples. CO?lClUSb~

Although not one optosensing approach is clearly superior to all the others in all analytical respects, the RTP detection principle presented here for glucose exhibits the typical advantages of phosphorescence measurements over the more conventional fluorescence mode, namely: in-

creased selectivity because not many substances phosphoresce and there is a time discrimination ability to remove fast emission phenomena (i.e., fluorescence, scattering, Raman, etc.) by using adequate “delay” times, and moreover the RTP sensor is not affected by the protein content in real samples; longer wavelength (less background) and wider separation between excitation and emission maxima are observed (emissions in the long-wavelength visible region allows us the use of cheap optical fibres and photodiode technology currently available); and the measurement of excited triplet state lifetimes &s-s> is much easier than that for singlet states (ns), hence multi-dimensional analysis with simple instrumentation and increased selectivity by resorting to chemometric treatments [27] can be envisaged. The RTP sensing principle proposed here can probably be extended to other enzymes involving molecular oxygen consumption (e.g., preliminary experiments with ascorbate oxidase were successful). Financial support from the Fondo para las Investigaciones Cientificas de la Seguridad Social (FISS) and the FundacGn para el Foment0 en Asturias de la Investigacibn Cientifica y T&nica (FICYT) is gratefully acknowledged. Y.M. Liu thanks the Spanish Education and Science Ministry for a Postdoctoral Fellowship.

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