J Sol-Gel Sci Technol (2009) 50:437–448 DOI 10.1007/s10971-009-1915-4
ORIGINAL PAPER
Fiber-optic glucose biosensor based on glucose oxidase immobilised in a silica gel matrix M. Portaccio Æ M. Lepore Æ B. Della Ventura Æ O. Stoilova Æ N. Manolova Æ I. Rashkov Æ D. G. Mita
Received: 30 October 2008 / Accepted: 2 February 2009 / Published online: 19 February 2009 Ó Springer Science+Business Media, LLC 2009
Abstract A monolithic silica gel matrix with entrapped glucose oxidase was constructed as a bioactive element in an optical biosensor for glucose determination. Physicochemical and biochemical characterizations of the catalytic matrix were performed, and the intrinsic fluorescence of immobilised glucose oxidase (GOD) was investigated in the UV and visible range by performing steady state and time course measurements. In all cases, the silica gel matrix proved to be a suitable support for optical biosensing owing to its superior optical properties (e.g., high transmittance and reliable fluorescence and GOD absorption spectra after immobilisation). From steady state measurements, calibration curves were obtained as a function of glucose concentration. When time course measurements were performed, the silica gel support displayed a larger linear calibration range and higher sensitivity than other immobilisation systems. In addition, a glucose optical biosensor was developed and characterised using as catalytic element GOD immobilised on a gel disk bound to a bundle of optical fibres.
M. Portaccio M. Lepore B. Della Ventura D. G. Mita Dipartimento di Medicina Sperimentale, Seconda Universita` di Napoli, Naples, Italy M. Portaccio M. Lepore B. Della Ventura D. G. Mita Consorzio Interuniversitario INBB, Sezione di Napoli, Naples, Italy O. Stoilova N. Manolova I. Rashkov Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria D. G. Mita (&) Institute of Genetics and Biophysics of CNR, Via Pietro Castellino, 111-80131 Naples, Italy e-mail:
[email protected]
Keywords Silica gel Glucose oxidase Glucose biosensing Optical biosensor
1 Introduction Sol–gel matrices have been increasingly utilised as sensing materials for optical biosensors owing to their superior chemical stability, optical transparency and porosity [1–3]. Biosensors, self-contained analytical devices able to detect specific molecules, are comprised of a sensing layer for selective molecule recognition and a transducer for converting physical–chemical changes associated with analyte recognition into detectable output [4]. Selective recognition in the sensing layer can be achieved by immobilising an appropriate affinity system such as enzymes or antibodies. Many biosensors are amperometric, utilising an enzymatic reaction to generate electrical output from the transducer. Optical sensors, another class of biosensors, offer many advantages compared to conventional amperometric biosensors [5]. Optical transducers can quantitatively determine fundamental optical characteristics of the sensing molecule (e.g., intensity and/or endogenous or exogenous fluorescence lifetimes [6, 7]) to quantise analyte concentration. Optical sensors have no electrical contacts with the sample and hence no signal interference from the reaction site to the detectors. Most importantly, optical sensors can achieve lower detection levels and can potentially be used for early detection of biochemical changes [8]. In these devices, inorganic alkoxides are usually used as the sensing layer because transduction requires a transparent glassy matrix. The sol–gel process offers a convenient and versatile method for preparing optically transparent matrices at room temperature [9]. The gel is produced in two steps:
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hydrolysis of an alkoxide precursor under acidic or basic conditions and subsequent condensation of hydroxylated monomers to create a porous matrix. Acidic or basic conditions are necessary because the hydrolysis reaction rate is pH dependent and the reaction proceeds slowly at pH 7.0. The condensation process occurs by hydrolytic or alcoholytic silanol group polymerization to form Si–O–Si bonds. The condensation process is also pH-dependent. Under basic conditions, condensation is very fast, proceeding via monomer addition to the Si–O- groups to create spherical particles and a continuous network. Under acidic conditions, the condensation rate decreases with increases in the degree of substitution and proceeds mainly by reaction of neutral species (Si–OH and protonated groups), creating mainly unbranched chains. These highly cross-linked porous materials can be used to entrap electroactive species or enzymes [3, 10]. Encapsulated biomolecules like enzymes or antibodies exhibit properties similar to those exhibited in aqueous solution. There are several reports describing encapsulation of biological molecules within sol–gel matrices [11–14]. Tatsu et al. [15] developed an amperometric biosensor for glucose determination in a flow system by entrapping glucose oxidase in a silica sol–gel matrix and monitoring the glucose levels with an oxygen electrode. Optical glucose biosensing has been researched by many groups [16–21], and gel matrices have been found to be particularly appealing for the development of optical fibre biosensors [22]. As a result, we began investigating this kind of immobilization procedure in conjunction with our previous biosensing and immobilization procedures [23–27]. Sensing sol–gel systems coupled with appropriate fluorescent probes have been used for glucose detection since 1994 [28–31]. Fluorescence spectroscopy is a very powerful analytical tool to study the effects of various physical–chemical parameters (pH, polarity, viscosity, concentration, solvent effects, excited state molecular processes, etc.) on molecules in solutions [32]. Most importantly, fluorescence spectroscopy can provide information from molecules present in very dilute concentrations within a heterogeneous mixture. In this paper, we report results obtained from a biosensor comprised of a monolithic silica sol–gel entrapping a glucose oxidase (GOD) as sensing element coupled to a fibre-optic transducer element. GOD intrinsic fluorescence spectra were analysed in the UV and visible region in presence of different glucose concentrations, and the corresponding calibration curves were obtained. In addition, time course measurements were acquired following the procedures presented in previous works [27, 33]. Physicochemical and biochemical characterizations of our sol–gel are also reported.
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2 Materials and methods 2.1 Materials All chemicals were purchased from Sigma (Sigma Italia, Milan, Italy), with the exception of tetramethoxysilane (TMOS) and glucose oxidase (GOD), which were purchased from Fluka (Fluka Italia, Milan, Italy). GOD is a typical flavoprotein. GOD from Aspergillus Niger (145 Umg-1) is a dimer with two tightly bound flavoadenine (FAD) molecules per dimer. In the UV region, GOD displays a maximum absorption at 275 nm and a maximum intrinsic fluorescence emission at 340 nm due to the tryptophan residues. As all flavoproteins do, GOD displays absorption maxima in the visible region at about 380 and 450 nm and an intrinsic fluorescence emission maximum at about 530 nm at pH 6.5. As previously reported [25, 27, 34], changes in the UV and visible fluorescence of free and immobilised GOD have been found during its interaction with glucose because oxidised and reduced flavines exhibit different fluorescence intensities. 2.2 Methods 2.2.1 Preparation of the catalytic sol–gel matrices Silica gel matrices were prepared by rapid mixing of 800 lL of solution A with 800 lL of solution B in a polymethylmethacrylate cuvette. Solution A was obtained by slowly mixing (for 1 h at 4 °C) TMOS (1,550 lL) with H2O (450 lL) plus 40 mM HCl (30 lL) for acidic hydrolysis or 40 mM NaOH (30 lL) for basic hydrolysis. Solution B contained 0.1 M phosphate buffer at different pH values (pH 5.5–7.5) depending on the desired condensation. The A and B solution mixture (1,600 lL) was poured into a polymethylmethacrylate cuvette that was sealed with paraffin film and placed horizontally until the gel formed. To avoid cracking once the gel was formed, the cuvette was filled with 0.1 M phosphate buffer at pH 6.5 and stored overnight in a refrigerator at 4 °C. The next day, the silica gel layer was removed from the cuvette and was ready to use. The catalytic gel was prepared via acidic hydrolysis, with solution B containing an additional 40 mg/mL of GOD in 0.1 M phosphate buffer at pH 6.5. For spectrofluorimeter measurements the resulting gel was a 8 9 35 9 3 mm parallelepiped in size, while a circular mould (3 mm in radius and 3 mm in thickness) was used as the bioactive element. The biosensor was constituted by a 0.5 m long Y-bundle (CeramOptec, GmbH, Germany) with 120 silica fibres in a circular arrangement.
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The bundle was split into two arms 0.2 m from one end, each containing half of fibres. All the ends were polished, and the bundle was covered with a flexible black insulator.
The same spectrophotometric assay was used to study the stability of the catalytic gel layer. For the absorption measurements, the experimental error never exceeded 4%.
2.2.2 Physicochemical and biochemical measurements
2.2.2.3 Biochemical characterization GOD activity was measured by determining the concentration of the produced hydrogen peroxide with Horseradish peroxidase (POD, EC 1.11.1.7) (1,280 U mg-1) [25] according to the following procedure: 50 lL of the sample to be analysed and 50 lL of POD (10 U mL-1) were added to 1 mL of 0.1 M acetate buffer at pH 5.0 containing 10 mM 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The ABTS concentration was in excess of H2O2 so that the reaction was limited by the H2O2 concentration. The reaction was allowed to proceed for 10 min at 25 °C until the H2O2 was consumed. A calibration curve of the absorbance as a function of H2O2 concentration gave an angular coefficient equal to 3 mM-1. Soluble GOD activity was measured according to the following procedure: 0.1 mL of GOD solution (154 U mL-1) was added to 1.9 mL of the D-glucose solution at the appropriate concentration, temperature and pH, so that the enzyme concentration was 7.7 U mL-1. Every 2 min, 0.1 mL of the reaction solution was taken out and added to 0.4 mL of a 0.1 M HCl aqueous solution to stop the enzyme reaction. The H2O2 concentration was then measured via the procedure described above. In this way, the H2O2 production as a function of time was obtained. The angular coefficient of the straight line fit of the initial H2O2 production gives enzyme activity measured in lmole min-1. To measure the activity of the catalytic gel samples, they were put in a reaction vessel filled with 20 mL glucose solution at the required concentration, temperature and pH. Then, 50 lL aliquots were extracted at regular time intervals and processed like the free enzyme. The angular coefficient of the straight line fitting H2O2 production as a function of time gives the activity of the catalytic membranes in lmole min-1. GOD activity measurements never exceeded 3% experimental error.
2.2.2.1 Diffusive permeability Since an enzymatic reaction rate depends on the substrate diffusion towards the catalytic site, we measured the diffusive permeability across our catalytic gels prepared under acidic or basic conditions using NaCl as a model diffusive species that does not interact with the enzyme. We determined the temporal changes in the electrical conductance in the two half-cells separated by a silica gel membrane and containing two NaCl solutions at different initial concentrations. A calibration curve of electrical conductance as a function of NaCl concentration allowed for NaCl concentration change measurements in the two half-cells of the reactor. Here, one half cell was initially filled with 1 M NaCl aqueous solution, while the other half cell was filled with double distilled water. The diffusion coefficient D (cm2 s-1) was calculated as follows: J¼
1 Dc Dc V ¼D A Dt Dx
ð1Þ
where J is the NaCl diffusive flux measured in mole/cm2 s, Dc/Dt is the concentration (mole/cm3) changes in the time Dt (s), A (cm2) is the surface area of a Dx (cm) thick gel membrane, and V (cm3) is the solution volume in each halfcell. Once D is calculated, one can estimate the values of the diffusive permeability coefficient P (cms-1) as follows: P¼k
D Dx
ð2Þ
where k is the partition coefficient between the bulk solution and the gel matrix. In the calculation, we set k = 1 whereas the molar ratio in the sol–gel was TMOS:H2O = 1:2.5. The experimental value of the diffusive permeability coefficient therefore contained a 2.4% average error.
2.2.3 Fluorescence measurements 2.2.2.2 Transmittance measurements The transmittance of silica gels, spectrophotometrically determined with a Lambda 25 Perkin Elmer spectrophotometer, was measured as the integral area under the spectrum in the range from 250 to 700 nm. For these measurements, the gel layer was placed in a cuvette filled with 3 mL 0.1 M phosphate buffer solution at pH 6.5. To verify the existence of structural changes as a result of immobilization, the absorption spectra of the free and immobilised GOD were compared. For the free enzyme, a GOD solution of 5 mg/mL in 0.1 M phosphate buffer at pH 6.5 was used.
2.2.3.1 Intrinsic fluorescence emission measurements The emission fluorescence spectra were collected with a spectrofluorimeter (Perkin–Elmer, model LS55) equipped with a Xenon discharge lamp with an emission spectrum ranging from 200 to 800 nm. In the UV range, samples were excited at 275 nm, while the emission spectrum was recorded between 310 and 410 nm. In the visible range, samples were excited at 450 nm and emission spectra were considered between 500 and 580 nm. Spectra were acquired with entrance and exit slits fixed at 5 nm with a 100 nms-1 scan speed.
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For analytical purposes, the emission fluorescence spectrum was treated as the size of the peak at 330 nm (530 nm) or as size of the integral area under the spectrum in the region 310–410 nm (500–580 nm) for the UV (visible) range. To test biosensor performance, we used a Xenon lamp source (Mod. PX2-Ocean Optics Ltd), a monochromator (Mod. USB 2000, Ocean Optics Ltd.) for selecting appropriate wavelengths and a photomultiplier tube (PMT) (Mod. R928, Hamamatsu, Japan).
present in the values of NaCl diffusion coefficients in the two different gel matrices; (2) in both gel systems, the values of the diffusion coefficients linearly decrease with pH increases in condensation process; and (3) the decrease rate for unit pH is 3.75 9 10-6 (cm2 s-1/pH) for acidic hydrolysis and 3.50 9 10-6 (cm2 s-1/pH) for basic hydrolysis. Since the rates are very similar, the same linear equation D ¼ 3:11 105 1:5 106 3:66 106 2:30 107 pH value ð3Þ
2.2.3.2 UV fluorescence time course measurements Changes in fluorescence measurements during the enzymatic reaction were followed with the above-mentioned spectrofluorimeter. As a baseline for subsequent measurements, the initial fluorescence emission of immobilised GOD at 330 nm was measured (excitation wavelength equal to 275 nm). After the addition of 200 lL glucose solution at different concentrations, changes in fluorescence intensity were monitored. As in Ref. [33, 34], the glucose concentration was determined by two different parameters: tapp and the linear slope of the intensity signal rise (dI/dt).
can be used for fitting all the experimental data with a high correlation factor (R = .99223). It is interesting to observe that our values for diffusion coefficients are of the same order of magnitude of those found in the literature for other gel systems [1].
3 Results and discussion 3.1 Diffusion coefficients In Fig. 1, the NaCl diffusion coefficients across the gels prepared under acidic (pH = 4.0, d) or basic (pH = 11.0, j) hydrolysis conditions are reported as a function of the pH used during the condensation process. From Fig. 1, it is clear that: (1) at each pH value used in the condensation process, no significant differences are
3.2 Transmittance measurements In Fig. 2, the transmittance of the gel prepared under acidic or basic hydrolysis conditions are reported as a function of the pH value used during the condensation process. Transmittance is expressed as the total area under the spectra from 250 to 700 nm. Figure 2 shows that: (1) at each pH value of the condensation process, the transmittance values of the gels prepared via acidic (pH = 4.0, d) hydrolysis are an order of magnitude higher than the values of those prepared via basic (pH = 11.0, j) hydrolysis; (2) the transmittance values for the gel prepared via acidic hydrolysis linearly decrease with increases in the pH of the condensation process, exhibiting an opposite trend to that of the gel prepared under basic hydrolysis; and (3) the rate of transmittance decrease with pH of the condensation process is
9
30.0
8
26.7
7
23.3
6
20.0
5
16.7
4
13.3
3
10.0
2
6.7
28500 28000 27500
3.3
1 5.5
6.0 6.5 7.0 pH in condensation process
7.5
Fig. 1 NaCl diffusion coefficients (left axis) or diffusive permeability (right axis) across different gel samples as a function of the pH used in the condensation process. Symbols for the pH hydrolysis conditions: d = pH 4.0; j = pH 11.0
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Transmittance (a.u.)
33.3
P ( 10-6 cm s-1)
10
Diffusive Permeability Coefficient
Diffusion Coefficient D ( 10-6 cm2 s-1)
29000
27000 26500 26000 4500 4000 3500 3000 2500 5.5
6.0 6.5 7.0 pH in condensation process
7.5
Fig. 2 Transmittance for different gels as a function of the pH used in the condensation process. Symbols for the pH hydrolysis conditions: d = pH 4.0; j = pH 11.0
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-850 units of area/unit of pH, while the rate of transmittance increase with pH of the condensation process is about ?600 units of area/unit of pH. From these data, it is evident that gels prepared via acidic hydrolysis are more suitable than those prepared via basic hydrolysis for the catalytic element in optical biosensors. To determine if the immobilization procedure affected enzyme structure, we examined the absorption spectra of the free and immobilised GOD from 250 to 700 nm. The results of this investigation are reported in Fig. 3, where the normalised absorption spectra for the free and immobilised GOD are reported as a function of wavelength. The spectra show a peak at 275 nm due to the absorbance of tryptophan residues and two peaks at 380 and 450 nm due to the absorbance of FAD. Since the shape and the position of the peaks remained unchanged, we concluded that the GOD structure does not change as a consequence of the immobilization procedure in the gel. The results of Fig. 3 are in agreement with other data in the literature [35] reporting that sol–gel immobilization prevents alterations in GOD optical properties. Following the procedure described above for absorption measurements, changes in the 275 nm peak height were taken as a measure of catalytic gel stability. In the inset of Fig. 3, peak height values are reported as a function of the time elapsed since gel preparation. The data in the inset show high catalytic stability of the gel since only 2% of the original value is lost after 60 days. 3.3 Biochemical characterization Enzyme activity is markedly affected by environmental conditions such as pH. Changes in optimum pH value 1.0 120 Peak Value (a.u.)
Absorbance (a.u.)
0.8 0.6
100 80 60
0.4
0
10
20
0.2
30 40 Time (days)
50
60
0.0 250
300
350
400
450
500
λ (nm)
Fig. 3 Absorption spectra of free (——) and immobilised (- - - -) GOD. The silica gel matrix was prepared via hydrolysis at pH 4.0 and condensation at pH 6.5. Inset: Example of the long-term stability of a catalytic gel matrix. The silica gel matrix was prepared by hydrolysis at pH 4.0 and condensation at pH 6.5
and the pH-activity profile of immobilised enzymes compared to free enzymes depend on the support charges. These changes are attributed to partition effects that, owing to electrostatic interactions with fixed charges on the support, result in different concentrations of charged species (e.g., substrate, products, hydrogen or hydroxyl ions etc.) in the microenvironment of the immobilised enzyme compared to the bulk solution. One of the main consequences of these partition effects is a shift in the optimum pH towards more alkaline or acidic values for negatively or positively charged matrices, respectively. In Fig. 4a, the relative activities of the free and immobilised GOD are reported as a function of pH. From Fig. 4a, it is evident that, in agreement with the findings in Ref. [36], free and immobilised enzyme have the same optimum pH value (pH 5.0). The profiles of the activity–pH curves are different, the one of the immobilised GOD is broader indicating a less marked pH dependence. The ‘‘optimum pH range’’ was defined as the range in which the relative activity was between 90 and 100%. The optimum pH range occurs at pH 4.2–5.8 for the free enzyme and at pH 4.4–6.2 for the immobilised enzyme. Enzyme activity temperature dependence displays a bell-shaped curve with an optimum activity. The curve for an immobilised enzyme can be broader, narrower or identical to that of the free enzyme, but optimum activity generally shifts towards higher temperatures, an indication of higher resistance to thermal deactivation. This resistance results from enzyme structure strengthening or protection by the immobilization procedure. In Fig. 4b, the temperature dependence of the relative activities for the soluble and insoluble GOD is reported. Maximum activity is seen at *30 °C for free GOD and at *45 =C for the insoluble form, indicating that the silica gel matrix protects the enzyme structure from inactivation. The ‘‘optimum temperature range’’ (i.e., the temperatures at which the relative enzyme activity is between 90 and 100%) occurs between 19 and 37 °C for soluble GOD and between 37 and 52 °C for immobilised GOD. These results agree with those reported by Yang et al. [37] for similar silica gel systems. In Fig. 4c, normalised reaction rates (enzyme reaction rate per mg of free or immobilised enzyme) are reported as a function of the glucose concentration for soluble and insoluble GOD. Both enzyme forms, as expected, exhibit a Michaelis–Menten behaviour characterised by the following kinetics parameters: Km = 2.5 mM and Vmax = 26.1 lmole min-1 mg-1 for the soluble GOD, and Km,app = 2.8 mM and Vmax,app = 3.8 lmole min-1 mg-1 for the insoluble GOD. These results agree with the observation that limitations on substrate diffusion towards the catalytic site introduced by the gel matrix reduce either the apparent
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3.4 Fluorescence measurements
(a)
3.4.1 Preliminary measurements
90 80 70 60 50 40 30 20 10 0 3
4
5
6
7
8
pH 110
(b)
90 80 70 60 50 40 30 20 10 0 10
20
30
40
50
60
Reaction Rate x 102 (µmole min-1 mg-1)
Temperature (°C)
(c) 25
Normalized Fluorescence Intensity (a.u.)
Relative Catalytic Activity (%)
100
Fluorescence measurements are reported in Fig. 5. Normalised fluorescence spectra for free and immobilised GOD are reported as a function of wavelength in the UV (5a) and visible (5b) range. The results in Fig. 5 clearly indicate that immobilization does not affect GOD optical properties. The spectra in Fig. 5a and b, in addition, confirm that the immobilization procedures here described produces biomaterials for effective optical sensing. Although some changes may occur in gel pore structures [38], they do not have macroscopic effects on the absorption and fluorescence properties of GOD. In fact, in comparison to free GOD, the immobilization procedure does not significantly alter UV and visible fluorescence spectra.
1.1
(a)
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2
20
310
320
330
340
350
360
λ (nm) 15
10
5
0 0
5
10 15 [glucose] (mM)
20
Fig. 4 Reaction rate of free (s—) and gel immobilised (h- - - -) GOD as a function of: pH (a); temperature (b); and glucose concentration (c). The silica gel matrix was prepared by hydrolysis at pH 4.0 and condensation at pH 6.5
Normalized Fluorescence Intensity (a.u.)
Relative Catalytic Activity (%)
100
1.1
(b)
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 480
values of Vmax or the apparent affinity of the enzyme towards the substrate, leading to increases in the apparent value of Km.
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500
520
540
560
580
λ (nm)
Fig. 5 Fluorescence spectra of free (——) and immobilised (- - - -) GOD in the UV (a) and visible (b) ranges. The silica gel matrix was prepared by hydrolysis at pH 4.0 and condensation at pH 6.5
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function of glucose concentration. The subscript ‘‘C’’ indicates the glucose concentration, while the superscript values in ‘‘UV’’ indicates the emission range. The PUV C Fig. 7a have been decreased by PUV = 550 arbitrary 0 units, i.e., the peak value of immobilised GOD in the absence of glucose (when C = 0). The experiment was conducted at 25 °C with glucose in 0.1 M acetate buffer solution at pH 5.0. The data in Fig. 7a show a Michaelis–Menten-type behaviour and are fit by an equation of the type:
(a)
600 500 400 300
(ii) 200
(i)
100
PUV C ¼ 300
320
340
360
380
400
420
λ (nm)
ð4Þ
where the subscript ‘‘sat’’ indicates the peak value at saturation, and KPUV is a ‘‘pseudo’’ Michaelis–Menten constant. A Lineweaver-Burk plot of the results reported in Fig. 7a allows us to derive the KPUV and PUV sat values reported in Table 1. As in Ref. [34], KPUV and PUV are the optokinetic sat parameters.
(b)
80
60
(ii) 180
40
(a)
160 140
(i)
20
0 500
520
540
560
580
λ (nm)
Peak value (a.u.)
Fluorescence intensity (a.u.)
100
PUV sat C KPUV þ C
120
120
100
100
Peak value (a.u.)
Fluorescence intensity (a.u.)
700
80 60 40
Fig. 6 Fluorescence emission spectra in a the UV and b the visible ranges for GOD entrapped in a silica gel in absence (curve i) and in presence (curve ii) of glucose (2 mM)
80 60 40 20 0
20
0
1
0 0
3
4
5
6
10
20 30 [glucose] (mM)
40
(b)
14000 12000
14000
10000
12000
Integral area value (a.u.)
Integral area value (a.u.)
It is interesting to note that no fluorescence signal was obtained from gel samples without immobilised GOD in either of the investigated wavelength ranges. Figure 6a and b show the emission fluorescence spectra for GOD entrapped in the sol–gel in the presence (2 mM) or in the absence of glucose in the UV region and visible regions, respectively. An increase in fluorescence is evident in the presence of glucose. We verified that the changes in the fluorescence were not due to loss of GOD by diffusion from the gel to the solution since no fluorescence was visible after removing the gel. The results in Fig. 6 show that: (a) the signals in the UV region are nearly one order of magnitude higher than those in the visible region; and (b) the presence of glucose increases the GOD fluorescence intensity.
2
[glucose] (mM)
8000 6000 4000 2000
10000 8000 6000 4000 2000 0 0
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[glucose] (mM)
0 0
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[glucose] (mM)
3.4.2 Calibration curves in the UV region In Fig. 7a, the peak intensities, PUV C , of the emission spectra of entrapped glucose oxidase are reported as a
Fig. 7 UV range: a Fluorescence emission peak as a function of glucose concentration. Inset: glucose calibration curve. b Fluorescence emission integral area as a function of glucose concentration. Inset: glucose calibration curve
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Table 1 Optokinetic parameters from fluorescence peak size analysis for free and immobilised GOD evaluated from UV and visible emission spectra measurements GOD form
Spectral range
Kp (mM)
Psat (a.u.)
Free
UV
1.87 ± 0.50
156 ± 11
Linear range (mM)
SP (a.u) (mM-1)
Ref
Up to 1.0
36.3 ± 0.9
[27]
Free
VIS
0.47 ± 0.10
46.8 ± 4.7
Up to 0.5
54 ± 2
[25]
Immobilised in gelatine
UV
27.8 ± 4.9
546 ± 37
Up to 30
10.7 ± 0.3
[27]
8.68 ± 1.61
Immobilised in gelatine
VIS
90.9 ± 6.2
Up to 8
5.6 ± 0.2
Immobilised in silica gel
UV
3.7 ± 7
183.7 ± 10.9
Up to 5
25.5 ± 1.5
Present paper
Immobilised in silica gel
VIS
25.9 ± 5.8
120.9 ± 11.5
Up to 20
3.05 ± .07
Present paper
The inset in Fig. 7a shows the range in which emission peak intensity and glucose concentration display a linear relationship (R = 0.99): UV PUV C ¼ SP C
UV where SUV A is the measurement sensitivity. SA is reported in Table 2 together with an extension of the linear range of the calibration curve.
ð5Þ
The value of the SUV P coefficient, representing the measurement sensitivity calculated from the inset in Fig. 7a, is reported in Table 1, together with the extension of the linear range of the calibration curve. In Fig. 7b, the integral area values, AUV C , of the intrinsic fluorescence emission spectra in the range 300–380 nm are reported as a function of glucose concentration. In Fig. 7b, AUV 0 = 23,836 arbitrary units (the value of the integral area of the GOD emission spectrum in the absence of glucose) have been subtracted from the AUV values. The experiC mental conditions were identical to those in Fig. 7a. Data are well fitted by a Michaelis–Menten relationship AUV C ¼
[25]
AUV sat C KAUV þ C
ð6Þ
where AUV sat indicates the integral area value at saturation and KAUV is a ‘‘pseudo’’ Michaelis–Menten constant. KAUV and AUV sat , obtained by the Lineweaver-Burk plot of the results in Fig. 7b, are reported in Table 2. In the inset in Fig. 7b, the range of integral area values that vary linearly with glucose concentration is reported. These data are well fitted (R = 0.98) by the linear equation: UV AUV C ¼ SA C
ð7Þ
3.4.3 Calibration curves in the visible region In Fig. 8a, the peak intensities PVIS C of the intrinsic fluorescence emission spectra in the visible region for GOD entrapped in a silica gel are reported as a function of the glucose concentration. The experimental points in the figure were obtained by subtracting the fluorescence peak intensity of immobilised GOD in the absence of glucose, i.e., PVIS = 62 arbitrary units. The experimental conditions 0 (temperature, pH, and buffer solution) were the same of Fig. 7. Michaelis–Menten behaviour is observed here as well. The inset in Fig. 8a shows the linear relationship between fluorescence peak intensity of entrapped GOD and VIS glucose concentration. The values of KPVIS , PVIS sat and SP , calculated with equations identical to (4) and (5), are reported in Table 1. In Fig. 8b, AVIS C was obtained by subtracting the integral area in the absence of glucose, i.e., AVIS = 27,890 arbitrary 0 units, from the measured values of the integral area in the presence of glucose. In the inset of Fig. 8b, we show the range in which the integral area of the emission spectrum varies linearly with glucose concentration. Inserting the data in Fig. 8b into equations similar to (6) and (7) proVIS duces the values of KAVIS , AVIS listed in Table 2. sat and SA
Table 2 Optokinetic parameters from fluorescence area integral analysis for free and immobilised GOD evaluated from UV and visible emission spectra measurements GOD form
Spectral range
KA (mM)
Asat (a.u)
Linear range (mM)
SA (a.u) (mM-1)
Ref
Free
UV
1.6 ± 0.5
11,880 ± 960
Up to 0.8
1,780 ± 29
[27]
Free
VIS
0.47 ± 0.15
2,962 ± 230
Up to 0.5
3,530 ± 57
[25]
Immobilised in gelatine
UV
29.5 ± 7.3
41,526 ± 4,033
Up to 30
808.5 ± 22.1
[27]
Immobilised in gelatine
VIS
8.68 ± 2.15
5,406 ± 520
Up to 8
355 ± 10
[25]
Immobilised in silica gel
UV
7.9 ± 2.1
15,570 ± 1,173
Up to 10
1,067 ± 61
Present paper
Immobilised in silica gel
VIS
23.5 ± 4.9
9,320 ± 807
Up to 20
252 ± 6
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(a) Fluorescence Intensity (a.u.)
60 70 60
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Peak value (a.u.)
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0
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30 40 [glucose] (mM)
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Integral area value (a.u.)
Integral area value (a.u.)
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3000 2000 1000
5000 4000 3000 2000 1000 0 0 2 4 6 8 10 12 14 16 18 20 22
[glucose] (mM)
0 0
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30 40 [glucose] (mM)
t app 0
100
200
300
400
500
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700
Time (s)
Fig. 9 Changes in fluorescence intensity as a function of time during the enzyme reaction for GOD entrapped in a silica gel matrix. Experimental conditions: kexc = 275 nm, kem = 330 nm, [glucose] = 12 mM in 0.1 M phosphate buffer, pH = 6.5
6000
4000
220
60
(b)
5000
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200
8 10 12 14 16 18 20
[glucose] (mM)
0 10
t1
10 0
0
260
50
60
Fig. 8 Visible range: a Fluorescence emission peak as a function of glucose concentration. Inset: glucose calibration curve. b Fluorescence emission integral area as a function of glucose concentration. Inset: glucose calibration curve
For comparison, Tables 1 and 2 also contain the values we previously obtained for free GOD and other gel systems [25, 27]. As seen in Tables 1 and 2, GOD immobilised on a silica gel shows higher sensitivity in the UV spectral range than in the visible range for both peak and area analysis. As expected, the extension of the calibration linear range is more accurate for measurements performed in the visible spectral range. These results confirm the general observation that extension of the linear calibration range and sensitivity are not directly proportional to each other. Conversely, the Kp or KA parameters are directly proportional to the extent of the linear range, as reported also in our previous papers [25, 27]. 3.4.4 Calibration curves through time course measurements In our previous work [27], we demonstrated that the method proposed by Sierra et al. [33] for free GOD was applicable to GOD immobilised in a gelatine membrane.
Here, we extend the method to GOD entrapped in a silica gel matrix. Figure 9 shows the UV fluorescence signal upon addition of 200 lL 10 mM glucose solution to the immobilised GOD (final glucose concentration = 0.2 mM). As is evident, the fluorescence intensity initially remains nearly constant at I0. After some time (referred as the ‘‘appearance time’’, tapp), the fluorescence intensity increases gradually to a constant value I1 at t1. Some time later, the fluorescence signal gradually decreases. We verified that the changes in fluorescence were not due to the presence of GOD in solution. According to references [27, 33], dynamic changes in the emission spectra can be used to determine glucose concentration by utilising two parameters: the linear slope Sl of rise in fluorescence intensity and the appearance time (tapp). The linear slope Sl is defined as dI/dt. Previously, we successfully used the parameter (tm - t0) in place of tapp, which is more difficult to determine. (tm - t0) is equal to the time required for the fluorescence intensity signal to reach a value equivalent to 10% of the overall increase. In the present study, we used both Sl and (tm - t0). Figure 10a reports the values of the linear slope of the fluorescence intensity increase in the UV region at different glucose concentrations. Again, Michaelis–Menten behaviour is observed, and the experimental values are fit by the curve represented by: Slc ¼
Slsat C KSl þ C
ð8Þ
where C, S1sat and KSl are defined as above. In the Fig. 10a inset, we report the linear calibration range for the experimental points in Fig. 10a. The linear relationship can be written as: Slc ¼ SSl C
ð9Þ
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determination. In particular, it was found that the linear calibration range depends on GOD concentration and the initial [O2]. In our case, [O2], measured with an oxygen electrode, was equal to 2.2 9 10-4 M. For our calibration curve therefore, we used glucose concentrations greater than 4.4 9 10-4 M. The linear range of the values of h i ½G (tm - t0) versus ln ½G2½ O at different glucose concen-
(a)
1,8 1,6
2,0
1,2 -1 Slope dI/dt (u.a.s )
Slope dI/dt (u.a.s-1)
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G=55mM
0.00
0.02
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0.08
trations is reported in Fig. 10b. This represents an alternate presentation of the data reported in Fig. 10a. Results in Fig. 10b indicate that, using this approach, the linear range in which the new calibration curve applies can be extended from 5 to 55 mM. The results also indicate that it should be feasible to use different GOD and oxygen concentrations to modulate the range in which accurate measurements of glucose concentrations can be obtained via time course fluorescence assays. Characteristic parameters for the time course approach (indicated TC) are reported in Table 4. STC (measured in seconds) is the slope of the best-fit line for the experimental values reported in Fig. 10b. From the data reported in Tables 3 and 4, it is clear that immobilization in silica gels effectively broadens the linear range for time course analysis compared to free and gelatine immobilised GOD. GOD immobilised in a silica gel also shows higher sensitivity than GOD immobilised in gelatine.
0.10
3.5 Design and validation of a fibre-optic biosensor
log([G]/([G]-2*O2]))
Fig. 10 a Slope of the rise in intensity of the fluorescence signal as a function of glucose concentration. Inset: h glucose i calibration curve. b
½G (tm-t0) values as a function of ln ½G2½ O2 at different glucose concentration
Our results with respect to linear calibration ranges and sensitivity confirm that GOD immobilised in our silica matrix performs similarly to GOD in other gel systems [37, 39–41]. For example, linear relationships are obtained up to 5–7 mM glucose with sensitivities around 7–10 (a.u./mM). Moreover, recent results [42] and our present measurements
where SSl is the measurement sensitivity. Values of Slsat, KSl and SSl are listed in Table 3. The model presented by Sierra et al. [33] predicts that (tm - to) is influenced by both the concentrations of oxygen [O2] and glucose [G]: (tm - to) varies linearly with h i ½G ln ½G2½ O : Obviously, this relationship is valid for glu-
Table 4 Optokinetic parameters for free and immobilised GOD obtained by fluorescence time course measurements and from (tm - t0) value analysis
cose concentrations greater than twice [O2]. The authors tested the model reliability with respect to enzyme and O2 concentrations, and their results confirmed that this approach could be used successfully for glucose
Immobilised in gelatine
GOD forms
STC (s)
2
Free Immobilised in silica gel
59 ± 8
TC Linear Ref range (mM) 0.6–1.2
[27]
337.8 ± 43.6 2.5–20
[27]
586 ± 81
5–55
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Table 3 Optokinetic parameters for free and immobilised GOD obtained by fluorescence time course measurements and by analysis of the slope of the rise in signal intensity GOD forms
KSl (s-1 mM)
Slsat (a.u.) (s-1)
SSl (a.u) (s-1 mM-1)
Linear range (mM)
Ref
Free
0.56 ± 0.10
97 ± 8
99.1 ± 3.9
Up to 0.6
[27]
Immobilised in gelatine
9.69 ± 2.38
0.44 ± 0.05
0.027 ± 0.001
Up to 10
[27]
Immobilised in silica gel
13.1 ± 2.9
2.1 ± .2
0.076 ± .003
Up to 20
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(inset in Fig. 3) on time-stability suggest that the sol–gel immobilization technique is able to retain enzyme activity longer than other conventional methods. Thus, we believe an inexpensive and easy-to-use biosensor could be constructed using optical fibres interfaced with a properly designed catalytic matrix. Figure 11 portrays a schematic of the set-up for testing the optical fiber biosensor utilising our catalytic silica matrix. The bioactive element contained a disk of our catalytic gel (6 mm in diameter and 3 mm in thickness) bound to a bundle of optical fibres with a screw cup. A detailed description of the Y bundle was given in Sect. 2.2. One end of the Y bundle was connected to the excitation source equipped with a monochromator, and the other end was connected to the detector. An optical filter (Melles-Griot, France) and a lens focusing system were placed between the end connected to the detector and the entrance to the PMT housing. The optical fibre bundle and detector allowed us to perform fluorescence measurements in the visible region. We excited the system at 450 nm, and the filter allowed us to detect fluorescence in the 500–580 nm range. The sensing end of the biosensor was in contact with a cuvette containing glucose solutions placed in a modified holder (Mod. CUV-ALLUV 4-way, Ocean Optics-Ltd). The detector output signals were sent to an oscilloscope (Mod. 123, Fluke). With this
set-up, we obtained a linear calibration from 0.2 to 10 mM glucose reported in Fig. 12 (experimental error *10%). The sensitivity was 89.06 ± 13.67 mV/mM-1. These results, similar to those reported in the literature for similar fibreoptic biosensors [6, 43], were obtained using intrinsic GOD fluorescence without any labelling procedures. Furthermore, the data in Tables 3 and 4 indicate that the implementation of time course measurements with appropriate detectors and electronic circuitry will allow us to fully exploit the advantages of sol–gel immobilization procedure investigated here.
4 Conclusions Physicochemical and biochemical characterization of GOD immobilised in monolithic silica gel supports were reported together with measurements of its intrinsic fluorescence in the UV and visible range in presence of different glucose concentrations. Changes in intrinsic fluorescence were used to obtain linear calibration curves for glucose concentration determination. The same approach was used for time course measurements. When time course measurements were considered, the silica support allowed us to obtain larger linear calibration ranges and higher sensitivity than those reported for other supports. Using the results of these investigations, a glucose optical biosensor was designed and tested. Acknowledgments This work was part of a collaboration between: the Italian Interuniversity Consortium ‘‘National Institute of Biostructures and Biosystems (INBB)’’, Rome, Italy and the Institute of Polymers, Bulgarian Academy of Sciences, and the Italian National Council of Researches (CNR) and the Bulgarian Academy of Sciences. I.R. acknowledges financial support from Grant TK-CH-1605.
Fig. 11 Fluorescence apparatus for fiber optic biosensor testing (S source, M monochromator, F optical filter, PMT photomultiplier, O oscilloscope, SC sample cell). Inset: biosensor schematic
Fluorescence Intensity (a.u.)
1000
800 600 400 200 0 0
2
4
6
8
10
[glucose] (mM)
Fig. 12 Glucose calibration curve obtained with the biosensor represented in this figure
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