Chapter 3. Results
3.4 Coupled enzyme assay to determine β-D(+)-glucose and histamine 24
Coupling glucose oxidase and diamine oxiadse catalyzed reaction with ABTS/H2O2 colorimetry system, we get the enzyme kinetic analysis of β-D(+)-glucose and histamine standard curves. All the curve and Km value are obtained with Michaelis-Menten equation in enzyme kinetics analysis of Sigma Plot 2001.
Enzyme kinetic analysis of glucose oxidase and diamine oxidase observed in our assembled biosensor system is shown in Fig. 22(a) and Fig. 23(a). By this process the Km value of β-D(+)-glucose and histamine were 4.9 ± 0.5 μM and 125 ± 3.21 μM, respectively, which approached to those obtained from UV/Vis spectrophotometer
(7.2 ± 0.2 μM and 160 ± 19.7 μM ). (See TABLE 3(a), TABLE3(b), Fig. 22(b) and
23(b)) The optimal reaction conditions of these two enzyme was showed in TABLE 2,
and the CMOS chip output voltage at different glucose and histamine concentrations were showed in Fig. 22(c) and Fig. 23(c). β-D(+)-glucose detection limit of assembled system is 1 μM and almost the same as that from UV/Vis spectrophotometer. On the other hand, the histamine detection limit of assembled system is 10 μM while the hitamine detection limit of UV-vis spectrophotometer is 20 μM. The liner detection ranges of our assembled biosensing system ranges from 1 μM to 5 mM and 10 ~ 80 μM, respectively.
CHAPTER 4. DISCUSSION Optimal absorptive wavelength
There are two factors to determine the optimal absorptive wavelength β-D(+)-glucose and histamine oxidation reactions: The absorption wavelength of the
products of β-D(+)-glucose and histamine oxidation reactions; and the absorption peak of CMOS photodiode chip. Owing to the maximum absorption peak of CMOS photodiode chip located between 600 and 700 nm and the absorption peaks of β-D(+)-glucose oxidation reaction were at 415 nm, 650 nm and 720 nm, the 650 nm
was chosen to be the optimal wavelength to prove the feasibility of our CMOS photodiode chip. On the other hand, the absorption peak of histamine oxidation reaction was only at 430 nm. Therefore, we follow the normal biochemical rule:
Chose the maximum absorption peak of reaction. As matter of fact, the only absorption peak of histamine oxidation reaction was at 430 nm based on the results of our experiment. Although 430nm might not the suitable range of the absorption peak of CMOS photodiode chip, we still wondered whether our CMOS photodiode chip could determinate histamine at 430 nm. Finally, we choose 650 nm and 430 nm to be the optimal absorptive wavelength of glucose and histamine oxidation reactions, respectively.
The performance of assembled biomedical system
Enzyme kinetic analysis results of glucose oxidase and diamine oxidase observed in our assembled biosensor system approached to that obtained from UV/Vis spectrophotometer. Our system was more superior than the traditional optical spectrophotometer whether volume or cost. In the meanwhile, the detection linear ranges of β-D(+)-glucose and histamine obtained from our assembled system approached to the amount in human bady. When we determine the β-D(+)-glucose and histamine. The liner detection ranges of our assembled biosensing system ranged from 1 μM to 5 mM and 10 ~ 80 μM, respectively, while the glucose concentration of human blood is 4 ~ 8 mM [30] and 1~8 mg/dl (90~720 μM) [31]. By the 2 to 10 folds sample dilution at most, the detection linear range of our assembled system can determine β-D(+)-glucose and histamine concentration in the human blood.
CHAPETR 5. CONCLUSION
We have set up an optical bio-sensing system for determination of glucose and histamine. A photodiode chip based on CMOS manufacture procedure was developed with the P+/Nwell finger structure and the subsequent current amplifier circuits, especially, include current mirrors and TIA. The photodiode with the P+/Nwell finger structure is surrounded by double guard rings to keep the substrate noise off. The optical transition theorem is used to be the detection method in our system. We designed colorimetrical reactions to be a biochemistry detection platform. Using the platform technology, we can determinate β-D(+)-glucose and histamine in the reagents. The performance of our system can compare with traditional large-size optical instrument, spectrophotometer. And our system has several advantages such as low cost, small size and the possibility of array-form development. In the future, we will aim to developing a new generation system based on this prototype system.
Minimization, array, user interface and micro-fluid channel would be integrated with the technology of the prototype system. Our hope is to set up a multi-functional household medical diagnosis instrument. And then, the research integrated with biochemistry and electronic engineering could benefit more people who need house medical care.
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TABLES AND FIGURES TABLEＩ
The growing global problem of diabetes
Stati 19 20
(e
202 (est
stics 95 00
stimate)
5 imate) People with diabetes (million) 135 154 300
% in developed countries 25.2 23.8 17.1
Female/male ratio 1.18 1.16 1.12
Soure: World Health Organisation.
It still gets worse that incidence of the disease has risen by an alarming 11% over the recent years, and a further doubling of new cases is predicted in the next 25 years.
TABLE II
Optimal conditions of enzyme reactions
glucose stamine hi
TABLE III Enzyme kinetic parameters
(a) glucose oxidase (GOx) catalysis reaction
(b) diamine oxidase (DAO) catalysis reaction
Enzyme kinetic analysis results of glucose oxidase and diamine oxidase observed
in our assembled biosensor system approached to that obtained from UV/Vis spectrophotometer.
TABLE IV
The feathers of spectrophotometer and the assembled biosensing system
Spectrophotometer Our biosensing system Feat 1. La
2. H (mor 3. H
(2.
1. Sm 2. Lo
(less than 15 thousand NTD) 3. High sensitivity
(3. ucose)
ures rge volume all volume
igh cost w cost
e than 300 thousand NTD) igh sensitivity
5 A650/min per uM of glucose) 0 A650/min per uM of gl
Figures
Biochemical reactions
Optical device Data process CMOS chip
Fig. 1. The flowchart of research. A biomedical sensor system assembled four
main components: optimal conditions of bio-reactions, CMOS chip consist of photodiode and circuit, optical devices, and data process software.
photodiode /
Fig. 2. The flowchart of CMOS optical biosensing system. A narrowband LED
430 nm system
was used as the light source, and a bandpass filter allowed the light of a specific wavelength to travel through the cuvette, in which the light was modulated by the biochemical materials. The light, after passing through the cuvette, was detected by the CMOS biochip. The CMOS photo detector converted the modulated optical signals into current signals, and then the transimpedance amplifier (TIA) converted them into voltage signals. Finally, the signals were recorded by a multimeter and processed by personal computer.
650 nm system
Fig. 3. Top view and cross section of P+/N well finger photodiode. Each P+
stripe is 1μm wide with 4 μm spacing, and the total area of this finger photodiode is 100 μm ×100 μm. The photodiode is surrounded by double guard rings to keep the substrate noise off; the P+ guard ring is connected to ground, and the N+ guard ring is connected to a high potential. Photodiode with finger structure (p+-n well) is designed by Yu-Wei Chang who is a Ph.D. student of the department of electronic engineering at NCTU.
Fig. 4. The configuration of subsequent circuits. The subsequent circuits include
a current source, current mirrors, a TIA, and output buffers. The TIA consists of a common-source stage (M0), two source followers (M1 and M2), and an external feedback resistance (RF). M1 serves in the feedback loop to isolate R0 from the loading effect, and M2 drives the load capacitance to alleviate the stability issue.
Current amplifier, TIA circuit with variable resistance are designed by Yu-Wei Chang who is a Ph.D. student of the department of electronic engineering at NCTU.
(a) (b)
Fig. 5. (a)The curve of LED (650nm) input voltage versus light intensity (b)The curve
of LED (650nm) input voltages versus CMOS chip output voltage (c). The curve of light intensity versus CMOS chip output voltage. With a 3.0 V power supply, the lower bound and upper bound of the output voltage are 0.49 V and 2.02 V, respectively.
(d) (e)
Fig. 5. (d)The curve of LED (430nm) input voltage versus light intensity. (e)The
curve of LED (430nm) input voltages versus CMOS chip output voltage (f). The curve of light intensity versus CMOS chip output voltage at 430nm.With a 3.0 V power supply, the lower bound and upper bound of the output voltage are 0.49 V and 2.02 V, separately.
Fig. 6. CMOS photodiode light absorption spectrum. It illustrates the
absorption peak of photodiode between 400 and 1000 nm, and it is obvious that the maximum absorption peak of photodiode within the visible light ranges between 600 and 700nm.
ABTS (36mM)
H2O2/ABTS/HRP→ABTS+
730 640
415
Fig. 7. The absorption spectrum (300~1000nm) of ABTS / H2O2 colorimetry
reaction. The scan parameters of UV-3310 are as follows: start wavelength is 1000
nm, end wavelength is 300 nm, delay time is 120 sec, scan speed is 300 nm/min and silt is 2 nm. Reaction condition is: 25 , pH 7.0, 100 mM ℃ Phosphate buffer, 30 mM ABTS, 0.2 mM H O2 2 and 0.2 unit/ml HRP. The absorption wavelength of the product
of reaction, ABTS
.
+has three absorption peaks at 415 nm, 640 nm and 730 nm, separately.
ABTS (36mM)
histamine/ DAO/ABTS/HRP→ ABTS.+
pH7.0 37℃
430nm
buffer
Fig. 8. The absorption spectrum (300~1000nm) of the coupled enzyme reactions
catalyzing histamine. The scan parameters of UV-3310 are as follows: start
wavelength is 1000 nm, end wavelength is 300 nm, delay time is 120 sec, scan speed is 300 nm/min and silt is 2 nm. Reaction conditions: 37 , pH 7.0, 100 mM ℃ Phosphate buffer, 30 mM ABTS, 0.4 mM histamine, 0.48 unit/ml DAO and 0.2 unit/ml HRP. The product of coupled enzyme reactions catalyzing histamine had only one light absorbance peak at 430 nm.
R2=0.9981
0.00 0.05 0.10 0.15 0.20 0.25 0
Fig. 9. The curve of initial rate versus the different HRP concentration Reaction
conditions: ABTS 30 mM, H2O2 400 μM, Phosphate buffer 100mM, pH 7.0 and 25 .℃ There is a linear range of HRP between 0.05 to 0.2 U/ml; therefore, we choose 0.2 U/ml of HRP to catalyze in the ABTS/H2O2 colorimetry system.
Michaelis-Menten
ABTS (mM)
Fig. 10. The curve of initial rate versus the concentration of ABTS. Reaction conditions : HRP 0.2U/ml, H2O2 400 μM, Phosphate buffer 100mM, pH 7.0 and 25 .℃
0 10 20 30 40 50
Initial rate (dA650/min)
0.0 0.5 1.0 1.5 2.0 2.5
Km=13.08 1.44 mM
±
GOx concentration determination
Fig. 11. The curve of initial rate versus the different GOx concentration
Reaction conditions: glucose 100mM, HRP 0.2U/ml, ABTS 30mM, phosphate buffer 100mM, pH 7.0 and 25 .℃ The linear range of glucose oxidase ranged from 0.01 to 0.8 U/ml.
DAO concentration determination
DAO (U/ml)
0.000 0.005 0.010 0.015 0.020 0.025
Fig. 12. The curve of initial rate versus the different DAO concentration
Reaction conditions: histamine 2mM, HRP 0.2U/ml, ABTS 30mM, phosphate buffer 100mM, pH 7.0 and 37 . The linear range of diamine oxidase ranged from ℃ 0.4 to 2 U/ml
Initial rate (dA430/min)
R2=0.9991
0.0 0.5 1.0 1.5 2.0 2.5
glucose couple enzyme reactions pH profile
2 3 4 5 6 pH 7 8 9 10
Initial rate (dA650/min)
0 1 2 3 4
Citrate buffer 100mM Phosphate buffer 100mM Tris buffer 100mM
Fig. 13. The pH profile of D-(+)-glucose coupled enzyme reactions. Reaction
conditions: glucose 100mM, HRP 0.2U/ml, GOx 0.48U/ml, ABTS 30mM, and 25 .℃
diamine oxidase pH profile
pH
6.4 6.6 6.8 7.0 7.2 7.4 7.6
Initial rate (dA430/min)
0.000 0.002 0.004 0.006 0.008
Fig. 14. The pH profile of histmaine coupled enzyme reactions. Reaction
conditions: histamine 400μM, HRP 0.2U/ml, DAO 0.8U/ml, ABTS 30mM and 37 .℃
Temperature effect of glucose couple enzyme reactions
Temperature (0C)
15 20 25 30 35 40 45
Initial rate (d650nm/min)
0.00 0.01 0.02 0.03 0.04 0.05 0.06
Fig. 15. The temperature effect of D-(+)-glucose coupled enzyme reactions.
Reaction conditions: glucose 100mM, HRP 0.2U/ml, GOx 0.48U/ml, ABTS 30mM, phosphate buffer 100mM and pH 7.0.
histamine-DAO temperature effect
Temperature ( 0C)
20 25 30 35 40 45 50
Initial rate (dA430/min)
0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040
Fig. 16. The temperature effect of hitamine coupled enzyme reactions. Reaction
conditions: histamine 2mM, HRP 0.2U/ml, DAO 0.8U/ml, ABTS 30mM, phosphate buffer 100mM and pH 7.0.
(a). 650 nm system
(b). 430 nm system
Fig. 17. The CMOS chip output voltage at different hydrogen dioxide
concentrations in the 650 nm and 430 nm system. Reaction conditions: (a)
HRP 0.2U/ml, ABTS 30mM, Phosphate 100mM, pH 7.0 and 37℃. (b) HRP 0.2U/ml, ABTS 30mM , Phosphate 100mM, pH 7.0 and 25℃.
(a). 650 nm bio-sensing system
H2O2 concentration curve
H2O2 (μM)
(b). 650 nm UV/Vis spectrophotometer
Fig. 18. Hydrogen dioxide detection limit in the 650 nm system. Reaction
conditions (both on (a) and (b)): HRP 0.2U/ml, ABTS 30mM, Phosphate 100mM, pH 7.0 and 25℃. (a) The detection limit is 1 μM. (b) The detection limit is 1 μM.
(a). 430 nm bio-sensing system
H2O2 concentration curve
H2O2 (μM)
(b). 430 nm UV/Vis spectrophotometer
Fig. 19. Hydrogen dioxide detection limit in the 430 nm system. Reaction
conditions (both on (a) and (b)): HRP 0.2U/ml, ABTS 30mM, Phosphate 100mM, pH 7.0 and 37℃. (a) The detection limit is 1 μM. (b) The detection limit is 1 μM.
H2O2 concentration curve
H2O2 (μM)
(a). 650 nm bio-sensing system
Hydrogen dioxide concentration curve (biosensor / 650 nm)
(b). 650 nm UV/Vis spectrophotometer
Fig. 20. Hydrogen dioxide linear detection range in the 650 nm system. Reaction
conditions (both on (a) and (b)): HRP 0.2U/ml , ABTS 30mM, Phosphate 100mM, pH 7.0 and 25℃. (a) Linear detection range：1 ~ 20μM. (b) Linear detection range：1 ~ 80 μM.
(a). 430 nm bio-sensing system
Hydrogen dioxide concentration curve ( biosensor system 430 nm)
H2O2 (μM)
0 5 10 15 20 25
Initial rate (dA430/min)
0.0 0.2 0.4 0.6 0.8 1.0 1.2
(b). 430 nm UV/Vis spectrophotometer
Fig. 21. Hydrogen dioxide linear detection range in the 430 nm system. Reaction
conditions (both on (a) and (b)):HRP 0.2U/ml, ABTS 30mM, Phosphate 100mM, pH 7.0 and 37℃. (a) Linear detection range：1 ~ 20μM. (b) Linear detection range：1 ~ 80 μM.
(a)
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.00
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.00
(c)
Fig. 22. Enzyme kinetic analysis curve of glucose oxidase. (a) on our assembled
biosensor system. (b) on UV/Vis spectrophotometer (UV-3310). (c) CMOS chip output voltage at different glucose concentration. Reaction conditions (on (a), (b) and (c)): HRP 0.2U/ml, GOx 0.48U/ml, ABTS 30mM, Phosphate buffer 100mM, pH 7.0 and 25 . Input voltage of CMOS chip is 3.0 V, and input voltage of LED is 2.2℃ 5V.
The detection limit are 1 μM on both (a) and (b). The liner detection ranges of our assembled biosensing system ranged from 1 μM to 5 mM on both (a) and (b).
(a)
0 200 400 600 800 1000 1200 1400
Initial r a te (dA430/min)
(c)
Fig. 23. Enzyme kinetic analysis curve of diamine oxidase. (a) on our assembled
biosensor system. (b) on UV/Vis spectrophotometer (UV-3310). (c) CMOS chip output voltage at different histamine concentration. Reaction conditions (on (a), (b) and (c)): HRP 0.2U/ml, DAO 0.8U/ml, ABTS 30mM, Phosphate buffer 100mM, pH 7.0 and 37 . Input voltage of CMOS chip is 3.0 V℃ and input voltage of LED is 3.5V. The detection limit is 10 μM on (a) and it is 20 μM on (b). The liner detection ranges of our assembled biosensing system range from 10 μM to 80 μM on (a) and 20 μM to 120 μM on (b).