Catalyzed determination of glucose with a mimic

Catalyzed determination of glucose with a mimic
enzyme constructed of β-cyclodextrin derivant∗
Shen Jingru
College of Chemistry and Life Science, South-Central University for Ethnic Communities,
Wuhan 430074, P. R. China
Abstract Catalyzed determination of glucose with mimic glucose oxidase was constructed by the
reaction of β-cyclodextrin, maleic anhydride, chloroactic acid with iron trichloride in hydrogen peroxide.
The method is simple and convenient, and sensitivity and reappearance are ideal. The linear range is
30~197µg/mL, while the detection limit is 4.10 µg/mL, the RSD is 0.98% (n=8). The recovery of
sample is 95.8%~103.1%.
Keywords Glucose, β-Cyclodextrin derivant, Mimic glucose oxidase, Catalysize to analyse
1 Introduction
Glucose determination is an important assay in clinical chemistry. The primary methods used are
chemical luminescence,[1] spectrophotometry,[2] and electrochemistry.[3] Although the methods of
measurement differ, the reactive mechanism (the concentration of glucose and oxygen to maltonic acid
and H2O2 by glucose oxidase) remains the same. The decrease in oxygen concentration or increase in
H2O2 concentration can be examined to determine glucose indirectly.[4] Because the enzyme reagent is
expensive, unstable when in contact with acid, alkaline, hot, rigorous under reactive conditions, and
prone to loss of activity and denaturalization, it is necessary to study the mimic bioenzyme.
Metalloporphyrin[5] has been used to mimic horseradish peroxide enzyme, coupling oxidate with
glucose oxidase (GOD) to measure glucose. In the present study, β-cyclodextrin derivant was utilized to
mimic GOD to determine glucose, adopting the method of o-toluidine.[6] The method is simple,
convenient, sensitive, and highly reappearance. Other parameters are as follows: Linear range, 30–197
µg/mL; detection limit, 4.10µg/mL; RSD, 0.98% (n=8), and sample recovery, 95.8–103.1%. Preparation
of the mimic enzyme is easy; it is inexpensive, reproducible, easy to be preserved, and not likely to be
metamorphic.
2 Experimental
2.1 Apparatus and reagents
The following apparatus/reagents were used: IR-440 infrared spectrophotometer, UV-240 UV-VIS
spectrophotometer, and AA-646 atomic absorption spectrum analyzer (Shimadzu, Tokyo, Japan);
752 UV-VIS spectrophotometer (Shanghai Exact Science Apparatus Ltd., Co. Shanghai, China); WH-1
miniwhirl mixer (Huxi Analytical Apparatus Factory, Sjanghai, China); pHS-3C acidometer (Wanda
Instrument Factory, Hangzhou, China); TG-332A microbalance (Analytical Apparatus Factory,
Shanghai, China); standard solution of glucose—0.1000 mol/L; N,N-dimethylformamide (DMF)
solution of o-toluidine, 0.4000 mol/L. All other reagents were of analytical reagent grade.
2.2 Synthesis of mimic enzyme
2.2.1 Preparation of compound 1.
β-cyclodextrin (CD) (11.300 g, 10 mmol) and maleic anhydride (3.200 g, 40 mmol) were crushed and
placed into a covered conical flask. The entire mixture was stirred thoroughly for ~4 hr at 80 °C until it
turned into a white solid. It was kept at this temperature for an additional 4 hr. Moderate acetone was
added, and the white precipitate was collected. After filtration of the precipitate, the white powder was
obtained. It is shown as compound 1 in Figure 1—weight, 9.400 g (7.1 mmol); yield, 71%. Elemental
∗
tel.: +86 27 87598575; fax: +86 27 67842123; e-mail: [email protected]. This project was supported by
the Natural Science Foundation of South-Central University for Ethnic Communities.
analysis produced the following results—C50H74O41•6H2O, computed value (%): C, 41.73; H, 6.02;
observed value (%): C, 41.95; H, 6.28. IR (KBr, cm–1); νmax: 3350 (s, OH), 2900 (w, CH2), 1720 (m,
C=O), 1620 (m, CH=CH), 1420 (w, COO–) cm–1. The 13C-NMR detection is shown in Table 1.
2.2.2 Synthesis of compound 2 in Figure 1.
Chloroacetic acid (0.800 g, 8.5 mmol) was placed into distilled water (40 mL). Compound 1 (2.700 g,
1.8 mmol) was then added and the mixture was stirred continuously for ~4 hr at 80 °C until the solution
turned slightly yellow. Moderate acetone was added, and the white precipitate was collected. After
filtration of the precipitate, it was washed with absolute alcohol until the C1– disappeared; it was then
dried. The product weight was 2.300 g (1.85 mmol) and overall yield was 75%. Because the product is
easily deliquated, 1 mol of compound 2 contains 10 mol of water. Elemental analysis produced the
following results: C54H82O45•10H2O, computed value (%): C, 39.75; H, 6.26; observed value (%): C,
39.49; H, 6.65; νmax: 3350 (s, OH), 2900 (w, CH2), 1720 (s, C=O), 1420 (w, COO-) cm–1. The 13C-NMR
detection of compound 2 is shown in Table 1.
Table 1
13
C NMR data of β-CD, compound (1) and compound (2) (D2O, 500MHz) (×10-6)
β-CD
Compound (1)
Compound (2)
δC
δc
δC
1(CH)
2(CH)
3(CH)
4(CH)
5(CH)
6(CH2)
104.42
74.63
75.63
83.68
74.36
62.83
O
β CD
+
HC C
HC C
104.43
74.64
75.65
83.68
74.37
62.82, 60.02
O
β CD O C CH =CHCOOH
O
O CH2COOH
β CD O C CH CH2COOH
H2O2
ClCH2 COOH
2
1
O
2
104.44
74.62
75.65
83.68
74.38
62.81, 60.02
O CH2COOH
β CD O C CH CH2COOH
FeCl3
. Fe
3+
2
2
3
O CH2COOH
β CD O C CH CH2COOH
. Fe . H O
3+
2
2
2
4
Figure 1 Mimic enzyme synthesis.
2.2.3 Synthesis of compound 3.
Forty milliliters of FeCl3 (0.500 mol/L) was poured into the 30mL (19.58 mol/L) liquid compound (2).
The color gradually changed to yellow and the solution became transparent. It was placed into a
thermostatic water bath at a temperature of 80 °C for 8 hr and was then filtered. Acetone was added to
the filtrate, and the faint yellow flocculent deposit was collected. It was washed with 95% ethanol until
no Cl– remained in the filtrate. The precipitate was placed into the desiccator; a dry, faint yellow, solid
compound (3), with a weight of 2.1 g and overall yield of 70%, was obtained. IR νmax: 3350 (s, OH),
2900 (w, CH2), 1720 (s, C=O), 1420 (w, COO–) cm–1. Atomic absorption spectrometry proved that
compound 2 S Fe3+ (mol ratio) = 1.09 in the coordination compound.
2.2.4 Synthesis of compound 4
Compound 3 (0.1000 g) was dissolved in 9.20 mL distilled water. To this was added 0.80 mL H2O2
(30%) while stirring continuously. After 30 min, compound 4 was obtained (Figure 1). The mimic
enzyme had a concentration of 6.556 mmol/L.
2.3 Catalyzed reaction of mimic enzyme
Compound 4 (4.00 mL) (6.556 mmol/L) was placed into the beaker with 2.00 mL distilled water, and
0.100 mol/L glucose (7.00 mL) was added while stirring continuously in a thermostatic water bath at 80
°C. Fifty microliters of the reaction solution was sampled by microinjection every 10 min. Next, 50 µL
(0.400 mol/L) o-toluidine was added and kept at 80 °C for 50minutes. Water was included to a fixed
volume of 5.0 mL, and the substrate (glucose) was replaced with water as the reference.
Using this method, A365 can be determined.[6] In experiment 1, compound 4 was replaced with
compound 3; in experiment 2, H2O2 replaced compound 4; and in experiment 3, FeCl3+ H2O2 substituted
compound 4. Other conditions were as above. The results are shown in Figure 2.
0.5
a
b
c
d
A
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
t (min)
Figure 2 Catalytic kinetics curve. System: Cmimic enzyme, 2.000 mmol/L; Co-toluidine, 0.2000
mol/L; temperature, 80 °C. a) β-CD-(M)2•Fe3+•H2O2. b) β-CD-(M)2 •Fe3+. c) H2O2. d).
FeCl3 + H2O2.
2.4 Glucose determination
To the covered, graduated tube (10.0 mL) was added a 2.000-mmol/L glucose solution, 6.556 mol/L
compound 4, and distilled water. The solution was mixed thoroughly and the reaction temperature was
set at 80 °C. After 50 minutes, 0.4000 mmol/L o-toluidine and water were added to a fixed volume of
5.0 mL. The substrate (glucose) was replaced with water as the reference. A365 can be determined under
these conditions.
3 Results and discussion
3.1 Affirmation of mimic enzyme method
As demonstrated in Figure 2, the catalyzed reaction rate using the mimic enzyme is faster than that by
FeCl3+H2O2, H2O2, and compound 3. The reaction rate of the mimic enzyme (compound 4) is rapid in
the first 0–40 min, and then the curve turns flat. This indicates that the reaction is complete.
3.2 Choice of reaction conditions
3.2.1 Influence of mimic enzyme concentration
To the covered, graduated tube (10.0 mL) was added 3 mL 2.000 mmol/L glucose solution, 6.556
mol/L compound 4 solution (50, 100, 120, 150, 200, 250, and 300 µL), and 2 mL distilled water. This
solution was mixed thoroughly and the reaction temperature was set to 80 °C. After 50 min, 200 µL
0.4000 mmol/L o-toluidine was added. After 1 hr, the substrate (glucose) was replaced with water as the
reference. A365 can be determined under these conditions.
As shown in Figure 3, A365 of 0.1311 mmol/L mimic enzymes is highest. The optimum molar ratio
between the mimic enzyme and glucose is 1S9.4.
0.8
A
0.7
0.6
0.5
0.4
0.3
5
10
15
20
25
30
35
40
c mimic enzyme (×10-2mmol•L-1);
Figure 3 Effect of mimic enzyme concentration on
sensitivity. System: Co-toluidine, 23.62 × 10–3 mmol/L;
temperature, 80 ˚C
0.8
A
0.7
0.6
0.5
0.4
0.3
0
5
10
15
20
-3
25
30
35
-1
C o-toluidine (×10 mmol·L )
Figure 4 Effect of o-toluidine concentration. System:
Cmimic enzyme, 0.1311 mmol/L; temperature, 80 ˚C.
3.2.2 Influence of o-toluidine concentration
Glucose solution (3 mL 2.000 mmol/ L) was placed into the covered, graduated tube (10.0 mL) along
with 100 µL 6.556 mol/L compound 4 solution and 2 mL distilled water. This was mixed thoroughly
and the reaction temperature was set to 80 °C. After 50 min, 0.4000 mmol/L o-toluidine (120, 160, 200,
240, 280, 320, 360, and 400 µL) was added. The substrate (glucose) was replaced with water as the
reference after 1 hr. A365 can be determined under these conditions. Figure 4 shows that the A365 of
23.62×10–3 mmol/L o-toluidine is highest, and A365 exhibits minimal change. The content of the
developer was saturated.
3.2.3 Effect of acidity
To the covered, graduated tube (10.0 mL) was added 1.50 mL 2.000 mmol/L glucose solution, 100 µL
6.556 mol/L compound 4 solution, and 3.50 mL buffer solution pH (5.40, 6.20, 7.70, 8.20, 8.80, 9.40,
and 10.0). This was mixed thoroughly and the reaction temperature was set to 80 °C; after 50 min,
0.4000 mmol/L o-toluidine 320 µL was added. After 1 hr, the corresponding buffer solution substituted
the substrate (glucose) as the reference. A365 can be determined under these conditions. Figure 5 shows
that the A365 of pH 8.83 is highest, and this is the optimum value of the reaction. It is possible that the
acidity affects the stability of the mimic enzyme structure. The optimum pH was 8.83.
0.4
A
0.3
0.2
0.1
5
7
9
11
pH
Figure 5 Effect of acidity. System: Cmimic enzyme, 0.1311
mmol/L; Co-toluidine, 23.62 × 10–3 mmol/L; temperature, 80 ˚C.
3.3 Standard curve and influence of interfering ion
Under optimum conditions (Cmimic enzyme, 0.1311 mmol/L; Co-toluidine,23.62 × 10–3 mmol/L; pH, 8.83;
temperature, 80 ˚C), A365 in the different glucose concentrations was determined. The standard curve is
linear when the concentration of glucose is 30–197 µg/mL. The standard regression equation is A = 2.4
× 10–3 C–0.0052; the standard regression coefficient is γ = 0.9994. Inorganic ions are contained within
the ampoule of the glucose. They are mainly interference ions. It is shown in the experiment that there is
no interference if the Zn2+ content is 70 times of glucose’s, when Cl–, K+, Ca2+, and Mg2+ are 100 times
of it, and when Ba2+ is 80 times of it. There is maximum interference if Al3+ is 60 times of glucose’s.
3.4 Precision
Using the above method, 35µL 2% glucose sample solution can be taken, A365 determined, and the
results calculated employing the standard regression equation. After an eight-time determination, the
average value is 140.6±1.365 µg/mL and RSD is 0.98%. According to the provisions of the International
Union for Pure and Applied Chemistry (IUPAC), the method detect ion limit is 4.095 g/mL.
3.5 Determination of sample and recovery experiment
As shown in Table 2, using the optimum standard curve conditions, the concentra-tion of glucose in a
5% ampoule can be determined and the results calculated utilizing the standard regression equation.
Table 2 Determination result and recovery of glucose in samples
Samples Found of Samples
Added
Total Found
Recovery
(µg•mL-1)
(µg•mL-1)
(µg•mL-1)
(%)
1
59.7
22.7
83.1
103.1
2
59.7
37.8
96.7
97.9
3
59.7
136.2
190.2
95.8
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