Impact of structural features of SBA-15 host particles on activity of
immobilized glucose oxidase enzyme and sensitivity of a glucose
sensor
Supplementary Information
Anees Y. Khan, Santosh B. Noronha* and Rajdip Bandyopadhyaya*
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
*
Corresponding authors.
Tel.: + 91 (22) 2576 7238; Fax: + 91 (22) 2572 6895, E-mail address: noronha@che.iitb.ac.in
(S.B. Noronha).
Tel.: + 91 (22) 2576 7209; Fax: + 91 (22) 2572 6895, E-mail address: rajdip@che.iitb.ac.in (R.
Bandyopadhyaya).
S1. Synthesis of porous silica particles
S1.1 Microporous silica (MS)
Synthesis protocol of MS was based on Onida et al. [1] and given as follows: TEOS is added to
aqueous solution of NaOH and reacted for 3 h at room temperature under constant magnetic
stirring. The molar ratios of the reactants are given below:
TEOS : NaOH : H2O = 1.00 : 0.40 : 525
Fig. S1. SEM images of MS particles (mean particle diameter 200 nm with standard deviation 25
nm).
Fig. S1 is an SEM image of MS particles. Its shows isolated particles with spherical
morphology. As MS particles prepared by above method cannot be analyzed in CLSM, due to
small particle diameter of 200 nm (Fig. S1), bigger MS were synthesized (mean diameter 35 μm,
with standard deviation 5 μm, Fig. 2a). Synthesis protocol was based on [2] and given as
follows: Acetic acid (AA, 11.4 ml) was added to 3.4 ml water under magnetic stirring followed
by addition of 10 ml of TEOS. The reaction was continued for 3 h at room temperature. The
molar ratio of reactants was as follows:
AA: H2O: TEOS = 1: 4: 4.
S1.2 Fibre-like SBA-15
Pluronic P123 (2 g) was dissolved in distilled water (65 ml) giving a concentration of 5.3
mM. Concentrated HCl (10 ml, 35%) was added and stirred for 4 h followed by drop-wise
addition of TEOS (4.5 ml) and stirred for 24 h at 35o C. The mixture was kept in a closed vessel
at 100o C for another 48 h under static condition. The molar ratio of reactants was as follows:
P123: HCl: H2O: TEOS = 1: 235: 11360: 60.
S1.3 Rod-like SBA-15
2.4 g Pluronic 123 was dissolved in 84 ml HCl solution (1.07 M) at 40o C giving a P123
concentration of 4.93 mM. Then 0.027 g NH4F was added followed by drop-wise addition of
5.466 ml TEOS under stirring (300-400 rpm) and reacted at 40o C for 20 h. The mixture was kept
in a closed vessel under static conditions for further reaction at 100o C for 48 h. The molar ratio
of reactants used was as follows:
P123: HCl: NH4F: H2O: TEOS = 1: 217: 1.8: 11030: 60.
S1.4 Cuboid SBA-15
1.5 g Pluronic 123 was dissolved in 55 ml of 1.3 M HCl solution at 30o C, giving a P123
concentration of 4.7 mM. Then 0.027 g NH4F was added followed by 11.762 ml decane and
stirred for 5 h. Finally, TEOS (3.464 ml) was added drop wise for 2 minutes. The mixture was
stirred at 30o C for 20 h and then kept in a closed vessel under static conditions for further
reaction at 100o C for 48 h. The molar ratio of reactants used was as follows:
P123: HCl: NH4F: C10H22 : H2O: TEOS = 1: 261:1.8: 135: 11278: 60.
In all cases, the solid product was filtered, washed, dried at ambient condition and
calcined at 540o C for 6 h.
S1.5 Morphological changes in SBA-15
A possible reason for these morphological changes in SBA-15 is as follows: on addition
of the silica precursor, TEOS, in an acidic medium, it undergoes hydrolysis and condensation
reactions resulting in polymeric silicate species. Then, self assembly between polymeric silicate
species and P123 micelles take place, which results in silicatropic liquid crystal seeds. These
seeds grow continuously via side-by-side anchoring along the long axis, and gives different
particle morphologies. Slower hydrolysis rate in the case of fibre (due to the absence of NH4F
being used as a hydrolysis-catalyst) may result in slower reaction and nucleation rate, hence
smaller number of initial seed particles, which continue to grow and yield larger fibre-shaped
particles at the end of the process.
NH4F was used as a hydrolysis-catalyst with mole ratio (NH4F/P123) from 0 (in case of
fibre) to 1.8 (in case of both rod and cuboid), which promotes polymerization of TEOS. The
latter condition may result in faster reaction and nucleation rates, and hence larger number of
seeds leading eventually to smaller rod- or cuboid-shaped particles. In addition, in case of cuboid
SBA-15 synthesis, decane serves as a pore expander, and also confines the silica doped micelles,
resulting in a decreased particle size [3].
S2. GOD Assay
A known mass of GOD immobilized SBA-15 was reacted with 100 mM glucose in 0.2 M
phosphate buffer (pH 7), for 10 min. at 26o C. Next, the supernatant was obtained on
centrifugation at 13000 rpm for 5 min. A known aliquot of the supernatant was mixed with an
assay mixture (containing 10 units of HRP, 0.2 mM AAP, and 2.5 mM phenol). The
concentration of the resultant pink quinoneimine complex, as determined spectrophotometrically
(at 510 nm) was obtained by multiplying the absorbance with the dilution factor and a calibration
plot. This gives activity of the immobilized GOD.
The step involving separate addition of the supernatant (which contains enzymatically
generated hydrogen peroxide product) to the assay mixture was required. There could otherwise
be an underestimation of the true activity due to adsorption of pink quinoneimine complex on
silica particles. Free GOD activity (in absence of silica) was measured under the same condition,
as that of immobilization conditions, for proper comparison. All experiments were carried out
under sterile conditions.
S3. Labeling of GOD with rhodamine red dye
A standard labeling procedure of rhodamine red dye on GOD was followed [4]. 10 mg of
GOD was dissolved in 1 ml of 0.1 M sodium bicarbonate buffer (pH 9). Separately, a dye
solution was prepared by dissolving 10 mg rhodamine red dye in 1 ml dimethyl sulfoxide. This
dye solution was then added slowly to the GOD solution and the reaction was continued for 4 h
under dark condition with constant stirring. Finally, the mixture was eluted through a PD-10
desalting column to separate out the rhodamine red labeled GOD with the unconjugated dye. The
concentration of dye labeled GOD was calculated to be 4.3 mg/ml with 1 degree of labeling. Dye
labeled GOD was then immobilized onto MS and fibre-like SBA-15 by a procedure as
mentioned in section 2.3.
S4. Materials characterization
S4.1 SAXS data
(100)
SBA-15 morphology
Fibre
Rod
Cuboid
(110)
(200)
Intensity (a.u.)
(110)
(200)
1.0
1.2
1.4
1.6
q (nm-1)
0.6
0.9
1.2
1.5
1.8
2.1
q(nm-1)
Figure S2. Small angle X-ray scattering (SAXS) of SBA-15 with fibre, rod and cuboid
morphologies. Based on position of the first peak, interpore distances for these morphologies are
10.6, 12.8 and 14.4 nm respectively. Inset shows SAXS for fibre and rod SBA-15.
For complementary confirmation of arrangement of mesopores, small angle X-ray
scattering (SAXS) data in Fig. S2 shows that SBA-15 with fibre and cuboid morphology have
three peaks which can be indexed closely to (1 0 0), (1 1 0) and (2 0 0) planes, respectively
[peaks corresponding to (1 1 0) and (2 0 0) planes for fibre-like SBA-15 is shown in inset]. The
rod form of SBA-15 has two peaks corresponding to (1 0 0) and (1 1 0) planes, respectively. This
confirms hexagonal arrangement of pores in all morphologies. Based on reflection of (1 0 0)
plane, the calculated interpore distances (for a hexagonal geometry) for fibre, rod and cuboid
SBA-15 are 10.6, 12.8 and 14.4 nm respectively.
S4.2 Nitrogen sorption data before and after GOD immobilization
(a)
600
Fibrelike SBA-15
Without GOD
With GOD
0.25
3
Pore volume (cm /g)
3
Quantity adsorbed (cm /g)
500
(b)
0.30
Fibrelike SBA-15
without GOD
with GOD
400
300
200
0.20
0.15
0.10
0.05
100
0.00
0
0.0
0.2
0.4
900
0.8
0
1.0
2
4
6
8
0.12
Rodlike SBA-15
Without GOD
With GOD
700
10
12
14
16
18
20
22
24
Pore diameter (nm)
(c)
800
(d)
Rodlike SBA-15
without GOD
with GOD
0.10
0.08
600
3
Pore volume (cm /g)
3
Quantity adsorbed (cm /g)
0.6
P/Po
500
400
300
200
0.06
0.04
0.02
100
0.00
0
0.0
0.2
0.4
0.6
0.8
0
1.0
P/Po
800
12
16
20
24
28
Pore diameter (nm)
0.08
Cuboid SBA-15
without GOD
with GOD
0.07
(f)
0.06
600
3
Pore volume (cm /g)
3
Quantity adsorbed (cm /g)
8
(e)
Cuboid SBA-15
without GOD
with GOD
700
4
500
400
300
200
0.05
0.04
0.03
0.02
0.01
100
0.00
0
0
0.0
0.2
0.4
P/Po
0.6
0.8
1.0
4
8
12
16
20
24
28
Pore diameter (nm)
Figure S3. (a) (c) (e) Nitrogen sorption isotherms; (b) (d) (f) pore diameter distributions based
on BJH model, for SBA-15 with fibre, rod and cuboid morphologies before and after GOD
immobilization, respectively. Immobilization conditions: GOD 0.45 mg/ml, 1.5 mg/ml of SBA15, 4o C, 0.2 M acetate buffer pH 4.0, immobilization time 96 h.
To get the specific surface area, pore diameter and pore diameter distribution of the
material, nitrogen sorption data were recorded. Fig. S3a, c, e show the nitrogen sorption
isotherms and Fig. S3b, d, f give pore diameter distributions for all the three morphologies,
before and after GOD immobilization (section 3.2). All samples exhibit type IV nitrogen
sorption isotherms, with H1 hysteresis loop, which is a characteristic of ordered mesopores, and
also confirmed by TEM images in Fig.1. The sharp inflections between the relative pressures
P/P0 = 0.60-0.9 in these isotherms correspond to capillary condensation within uniform
mesopores. Fig. S3e shows that cuboid SBA-15 has a larger mean pore diameter than other
samples.
S4.3 Nitrogen adsorption-desorption data for MS
25
20
15
Pore volume (cm3/g)
Quantity of nitrogen adsorbed (cm3/g)
0.008
10
0.006
0.004
0.002
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Pore diameter (nm)
5
0
0.0
0.2
0.4
0.6
0.8
1.0
P/Po
Figure S4. Nitrogen sorption isotherm for MS.
Nitrogen sorption isotherm of MS in Fig. S4 shows isotherm of type II, which is a characteristic
for a microporous material. Inset shows pore volume distribution. The BET specific surface area
for MS is 8.8 m2/g with pore diameter of 1.8 nm.
S4.4 Microporous nature of silica wall confirmed by BET data
0.016
Fibre
Rod
Cuboid
3
Pore volume(cm /g)
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Pore diameter (nm)
Figure S5. Micropore distribution in the wall of SBA-15 samples, based on Horvath-Kawazoe
method.
Fig. S5 shows that all morphologies of SBA-15 have microporous silica wall with a micropore
value of 0.8 nm.
S5. GOD immobilization and activity
S5.1 The reason for the background fluorescence in MS particles
The objective in Fig. 2a was to show that rhodamine labeled GOD (R-GOD) is
immobilized on the external surface of MS particles. As MS particles have very small external
surface area, R-GOD loading was very small. Therefore, removal of the excess unadsorbed RGOD during washing resulted in removal of loosely adsorbed R-GOD too. This further
decreased R-GOD loading and the sample did not show any fluorescence.
Therefore, the unwashed sample analyzed in Fig. 2a. shows fluorescence from the
external surface of MS particles and the background fluorescence (due to unadsorbed R-GOD)
from interstitial spaces between MS particles, but no fluorescence from within particles. This
confirmed presence of R-GOD on the external surface of MS particles.
In contrast, fibre-like SBA-15 particles have much higher specific surface area, hence
much higher R-GOD loading is obtained on the internal surface compared to that on the external
surface of MS particles. Subsequently, R-GOD immobilized fibre-like SBA-15 particles were
washed off to remove unadsorbed and loosely adsorbed R-GOD, and thus, a confocal image
(Fig. 2b) was obtained, showing a uniform fluorescence from within the particle, without the
background fluorescence. This confirmed that GOD gets immobilized inside pores of SBA-15.
S5.2 Presence of GOD inside mesopores confirmed by CLSM
The particle diameter of fibre-like SBA-15 is 4-5 μm (Fig. 1a), therefore, the length of the focal
plane was selected to be of 7.1 μm, which was divided into 29 optical slices each with 0.24 μm
length of the focal plane (Fig. S6). In first few optical slices (labelled as 0.24-1.96 μm in Fig.
S6), the external surface of the particle appears. In the middle of focal plane (2.2-3.91 μm), the
centre of the particle appears, afterwards, the particle starts to disappear (4.16-7.1 μm). This can
be observed by a slight change in the particle morphology. The particle morphology does not
change inside the particle, as can be seen in labels 2.2-3.91 μm of Fig. S6. Therefore, presence of
the fluorescence in the centre of the particle confirms that GOD is present in the centre of
particles. Hence, it can be inferred that GOD is present inside the mesopores of fibre-like SBA15. (The green fluorescence is due to labelling of GOD with FITC dye, which gives green
fluorescence).
Figure S6. Confocal laser scanning microscopy images of FITC-GOD conjugate immobilized in
fibrelike SBA-15. Conditions: FITC labelled GOD 1 mg/ml, silica 5 mg/ml, immobilization
temperature 4 oC, 0.2 M acetate buffer at pH 4, immobilization time 96 h.
S5.2 Oppositely charged GOD and silica species at pH 4.0
Fig. S7 shows the zeta potential measurement for GOD and SBA-15, with spherical MS as a
control sample. Under the experimental condition, GOD molecules acquire a positive surface
potential of +8.2 mV, whereas silica particles acquire a negative surface potential of –4.5 to –9.9
mV. Hence, due to electrostatic attraction, positively charged GOD molecules get physically
adsorbed onto the negatively charged silica surface.
Zeta potential (mV)
15
GOD
10
5
0
-5
Fibrelike
SBA-15
Rodlike
SBA-15
-10
Cuboid
SBA-15
MS
Figure S7. Zeta potential measured in 0.2 M acetate buffer pH 4.0, showing that immobilization
is favorable because of electrostatic attraction in between oppositely charge species. Conditions:
GOD 0.5 mg/ml, silica 0.5 mg/ml, 25o C, 0.2 M acetate buffer pH 4.0.
S5.3 Kinetic data of GOD immobilization
For GOD immobilization kinetics, the procedure was based on Lei et al. [5]. In a typical
experiment, SBA-15 was added to the GOD solution (0.2 M, pH 4.0 acetate buffer) in a covered
vessel to prevent evaporation. The mixture was stirred at 4o C for 28 – 38 h. The supernatant was
separated from the solid materials, by centrifugation at 15870 g for 10 minutes. The enzyme
content of the supernatant was measured in a spectrophotometer, using UV absorption at 280 nm.
The amount of GOD immobilized was calculated based on difference in enzyme concentration
before and after adsorption. Bradford’s assay [6] was used for detecting very small (up to 2
µg/ml at the minimum) GOD concentrations. This is a colorimetric assay for protein
quantification, based on shift of the Coomassie dye absorption maximum from 470 nm to 595
nm on binding to protein at acidic pH. Typically, 100 µl of GOD solution was mixed with 1 ml
of Bradford’s reagent, followed by 5 min incubation at room temperature and absorbance
measurement at 595 nm.
Fig. S8 shows that after 35 h of contact time, SBA-15 adsorbs approximately 37-50%
GOD, in comparison to only ~5 wt.% by MS. This clearly shows the superiority of SBA-15 over
MS, for GOD immobilization. The initial data (shown in inset) suggests that fibre and rod
morphologies adsorbs only 11.5 and 11.3 wt.%, respectively, whereas cuboid morphology
adsorbs up to 33.8 wt.%. This showed that the immobilization kinetics for fibre and rod SBA-15
is independent of as aspect ratio. This can be due to the molecular size of GOD (6.0 nm × 5.2 nm
× 7.7 nm [43]), being comparable to 6.8 nm pore, resulting in a larger diffusional resistance to
GOD molecules. In contrast, Fan et al. [7] reported that a decreased aspect ratio of SBA-15
resulting in a faster immobilization rate of lysozyme. This is obvious as the size of a lysozyme
molecule (3 nm × 3 nm × 4.5 nm [8]) is much smaller than pore diameter (~ 8 nm). An increase
in pore diameter from 6.8 to 11.4 nm with a 4 fold decrease in aspect ratio from rod to cuboid
SBA-15 resulted in a faster immobilization rate, by adsorbing approximately 3 times higher
GOD during the initial 30 min. of experiment. However, MS could adsorb only ~5wt% GOD.
This is because GOD can access only the small external surface area (8.8 m2/g).
100
80
GOD immobilized (wt%)
40
30
20
10
0
Microporous silica
Fibrelike SBA-15
Rodlike SBA-15
Cuboid SBA-15
90
70
60
0 1 2 3 4 5
50
40
30
20
10
0
0
5
10
15
20
25
30
35
40
Time (h)
Figure S8. GOD immobilization in microporous silica (MS) and SBA-15. Immobilization
conditions: GOD 0.42 mg/ml; silica 1.38 mg/ml; 4o C; 0.2 M acetate buffer pH 4.0. Inset shows
immobilization rates during initial hours of experiment.
S5.4 NAA at smaller Gld
(g free GOD/m2 silica)
Normalized absolute activity
4
Microporous silica
Fibrelike SBA-15
Rodlike SBA-15
Cuboid SBA-15
3
2
1
0
0
20
40
GOD loading density (g/m2)
Figure S9. Variation in NAA at smaller Gld
Fig.S9 shows that for small Gld values, NAA increases. It further shows that with increase in host
pore diameter, NAA increases.
S6. GOD does not leach in solution and structure of SBA-15 is robust
Immobilization is expected to be due to weak electrostatic attraction and consequently
more GOD can leach out from the cuboid form, as it has a pore diameter larger than that of the
GOD molecule. To evaluate it, after each cycle of use, the particles were washed two times with
phosphate buffer of pH 7, and centrifuged at 13000 rpm for 10 min to recover the particles.
Following this, the supernatant was analyzed in two ways: (i) Bradford assay can detect a
minimum of 2 µg/ml of GOD. (ii) Activity measurement of GOD can detect a minimum of 0.3
µg/ml of GOD. However, GOD was found to be below the detection limit using both the assays,
hence its loss by leaching was ruled out.
The final possibility of GOD leaching due to structural collapse of SBA-15 was also
considered. There are number of reports in the literature [9][10] which describe superior
hydrothermal stability of SBA-15 than MCM-41 due to its thicker pore walls (3.5-6 nm [S10])
than that of MCM-41 (wall thickness 2-2.4 nm [12]).As all SBA-15 morphologies had thick pore
walls (Table 2), they were expected to be structurally stable. Even if the structure collapse is
assumed to be one of the reasons for loss of GOD and RA, then the supernatant can detect that
GOD up to 0.3 µg/ml using activity assay (whereas the immobilized GOD amount was varied
from ~100-190 µg/ml). Since during reusability study, the leached GOD was found to be below
the detection limit, hence possibility of GOD leaching due to structural collapse was also ruled
out.
S7. Calibration of the glucose sensor
24
with glucose
without glucose (blank)
22
20
2
I (A/cm )
18
16
14
12
10
8
0
100
200
300
400
500
600
700
Time (s)
Figure S10. Current-time response data for working electrode modified with GOD immobilized
in cuboid SBA-15 Glde 1.1 µg/cm2 with ferrocene. Conditions: 20 ml of nitrogen saturated 0.2 M
phosphate buffer pH 7 under moderate stirring (80-100 rpm), temperature 27 oC, applied
potential + 0.5 V vs. SCE. Red curve is for blank while black curve is the current time response
in presence of glucose.
Figure S10 shows current-time data for (i) a blank run (without glucose aliquots) shown
as red curve, and (ii) test run (with glucose aliquots) shown as black curve. The current obtained
in blank run is called as the background current (or charging current). This current was then
subtracted from the test run, to generate a glucose calibration plot.
S8. Discussion on glucose sensing results
A comparison of the results from the present work with those of Wang et al. [13] is
reported in Table 2. They had used a GCE, modified with GOD immobilized in 7.4 nm SBA-15
pores at an estimated Gld of 0.09 µg/cm2. Linearity obtained was 0.5-6.0 mM glucose
concentration, at a relatively higher potential of +0.8 V (vs. Ag/AgCl), under deoxygenated
condition (sensor no. 1 in Table 2). Detection at a higher potential is generally avoided as it may
cause interference from some related biomolecules by oxidation, such as uric acid and dopamine
[14]. Therefore, compared to Wang et al. [13], the larger glucose sensing range (of 2.0-7.7 mM)
with a response time of ~ 30 s from the present work (that too at a lower potential of + 0.4 V vs.
SCE), may be attributed to increased pore diameter (11.4 nm) and small particle length (300 nm).
The latter results in more facile diffusion to both glucose and hydrogen peroxide molecules
inside and outside the mesopores, respectively, in relation to the bulk aqueous phase.
For glucose sensing, Zhou et al. [15] had also immobilized GOD in both rod and
spherical shaped SBA-15, with much higher Gld (sensor no. 2 and 3 in Table 2, respectively).
They obtained a linearity range of glucose of 0.1-7.5 mM for both cases, at an applied potential
of +0.6 V, with sensitivity of 1.6 and 2.42 µAcm-2mM-1, respectively. Their linear sensing range
was comparable to that reported in the present work (2.0-7.7 mM) at 0.1 µg/m2 for GOD-cSBAGCE. However, the latter has a poor sensitivity (0.34 nAcm-2mM-1) which is given by the slope
of the calibration curve. Higher sensitivity is desirable, whereby two close concentrations are
resolvable at higher current densities.
Sensor no. 4 in Table 2 enlists the result for Gld of 5.2 µg/cm2, as it showed the best
performance among all sensors made in the present work, achieving the comparable glucose
sensing range of all GOD-SBA-15 reported in literature. Cuboid morphology of SBA-15 of the
present work has pore diameter of 11.4 nm, with a smaller mesopore length and aspect ratio of
300 nm and 1.1, respectively. These result in much faster accessibility of glucose molecules to
interact with immobilized GOD. It translates into higher enzymatic activity and leads to a higher
rate of production of hydrogen peroxide and its facile outward diffusion from mesopores,
resulting eventually in a short response time. Finally, glucose sensing performance for cuboid
and rod morphology of SBA-15 of the present work was compared, in order to examine the role
of higher activity of cuboid over rod-shaped SBA-15 (Fig. 3b). The latter had a shorter mesopore
length of 580 nm (in comparison to 1.2 µm used by Zhou et al.) [15]. A glucose sensor with rodlike SBA-15 was made at 6.2 µg/cm2 and the sensing results are shown in Fig. 5d and reported as
sensor no. 5 in Table 2. It showed a linearity range of 2.0-7.7 mM, which is better than Wang et
al. [13], and comparable to Zhou et al. [15] However, comparing it with that of cuboid at Gld of
5.2 µg/cm2 clearly suggests that, indeed cuboid morphology is better than that of rod for glucose
sensing, due to the inherent difference in GOD activity.
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