0 5 10 15 20 25 30
1:6 1:9 1:12 1:15 1:18
Ratio
Current (nA)
Fresh 3Days
Figure 1.3 Response of glucose oxidase immobiled by PVA-SbQ photopolymerization on the carbon paste electrode. The ratio of GOx to ddH2O was 1:6, 1:9, 1:12, 1:15 and 1:18, respectively. The enzyme solution was prepared by mixing 10 mg GOx solution and 10 mg PVA-SbQ. The PVA-SbQ/GOx electrode is treated with 10 mM glucose under the fixed potential of 500 mV in the phosphate buffer, pH 7.0, at 37
℃.Stability of the GOx was operated under the same condition after three days.
0 5 10 15 20 25 30 35 40 45 50
0 100 200 300 400
Time(sec)
Current(uA)
0-2V 0-1.5V 0-1V
Figure 1.4 Response of electrodes modified by polypyrrole with different voltage ranges. The PVA-SbQ/GOx on Ppy-modified electrode is treated with 1 mM glucose under the 500 mV in a 50 mM sodium phosphate buffer, pH 7.0, at 37 ℃.
(a) (b)
(c) (d)
(e)
Figure 1.5 Cyclic voltammogram of pyrrole electropolymerization
under different ionic strengths Electropolymerization of pyrrole was performed in water containing 50 mM pyrrole and 100 mM (a), 250 mM (b), 500 mM (c), 750 mM (d) or 1000 mM (e) KCl using cyclic voltammetry. The cyclic voltammetry was carried out five cycles under the room temperature between the working potentials of 0.0 and 0.1 V at a scan rate of 50 mV/s.
0
Figure 1.6 Electrochemical responses of electrodes modified by polypyrrole. Various PVA-SbQ/GOx/Ppy-CPE biosensors was tested using 1 mM glucose as analyte at 37 ℃ in a pH 7.0 phosphate buffer.
The working potential was set at 500 mV relative to the Ag/AgCl reference electrode. The activity of the fabricated glucose biosensor, PVA-SbQ/GOx/Ppy-CPE, was operated under the same condition after five days to study its long-term stability.
0
(a)
(b)
Figure 1.7 SEM of polypyrrole-modified carbon-paste electrode. (a)
Surface of the bare carbon paste electrode. (b) Surface image of polypyrrole-modified carbon-paste electrode observed under the scanning electronic microscope. The polypyrrole was generated by electropolymerization in water containing 50 mM pyrrole in 100 mM KCl as mentioned in Fig. 1.5.
(a) (b)
(c) (d)
Figure 1.8 SEM of the polypyrrole-modified carbon-paste electrodes.
Surface images of the polypyrrole-modified carbon-paste electrodes were observed under the scanning electronic microscope. The polypyrrole was generated by electropolymerization in water containing 50 mM pyrrole and 250 mM KCl (a), 500 mM KCl (b), 750 mM KCl (c) or 1000 mM KCl (d) as mentioned in Fig. 1.5.
0 Figure 1.9 Repeat response of the electrode that modified using
polypyrrole in the (a) 750 mM KCl (b) 1000 mM KCl. The PVA-SbQ/GOx/Ppy-CPE biosensor was assayed using 1 mM glucose at 37 ℃ in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode.
(a)
(b)
Figure 1.10 SEM of the polypyrrole-modified carbon-paste electrode after treating with H2O2. The surface images of the Pd-modified carbon-paste electrode of untreated (a) and after treating three times with 1 mM H2O2 (b).
carbon paste electrode 50 nm
Figure 2.1 Cyclic Voltammograms of electrodes modified with different thickness of Pd. Solid line (▬) is the carbon-paste electrode modified with 50 nm Pd. Solid triangle (▲) represents the carbon-paste electrode modified with 100 nm Pd. Dash line (…) is the carbon-paste electrode modified with 150 nm Pd. Circle (○) is the bare carbon paste electrode.
0 100 200 300 400 500 600
0 0.2 0.4 0.6 0.8 1
Voltage (V)
Current (nA)
carbon-paste Sputtering (100 nm)
Figure 2.2 Hydrodynamic voltammogram of CPE and PdS100-CPE.
The electrochemical property of CPE (■) and PdS100-CPE (●) were studied using cyclic voltammetry in the presence of 1 mM H2O2, in 1 X phosphate buffer, pH 7.0.
0 1 2 3 4 5 6 7
0 100 200 300 400 500
Time(sec)
Current (uA)
50nm Pd 100nm Pd 150nm Pd
Figure 2.3 Response of the PVA-SbQ/Gox/PdS-CPEs. The
PVA-SbQ/Gox/PdS50-CPE, PVA-SbQ/Gox/PdS100-CPE and PVA-SbQ/Gox/PdS150-CPE were characterized for its response to 1 mM
glucose at 37 ℃ in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode.
Figure 2.4 Cyclic voltammograms of a carbon-paste electrode during
electrodeposition of Pd. The electrolyte was prepared as 5 mM palladium chloride in 1X PBS. The Pd electrodeposition was performed using cyclic voltammetry in a potential range from -0.4 to 1.0 V at the scan rate of 50 mV/s for 10 cycles.
0 1000 2000 3000 4000 5000 6000
10 cycles 15 cycles 20 cycles Sputtering 100 nm Pd
CPEs
Current (nA)
Figure 2.5 Responses of the different palladium-modified electrode.
The PVA-SbQ/GOx/Pde10-CPE, PVA-SbQ/GOx/Pde15-CPE and PVA-SbQ/GOx/Pde20-CPE biosensors were investigated for their responses to 1 mM glucose at 37 ℃ in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode.
0 50 100 150 200 250 300 350 400 450 500
0 50 100 150 200 250 300 350 400
Time(sec)
Current(nA)
Figure 2.6 Successive responses of the PVA-SbQ/GOx/PdS100-CPE
biosensor. The successive responses of fabricated PVA-SbQ/GOx/PdS100-CPE biosensor was characterized by step-wisely adding 10 µM glucose in the reaction chamber. The oxidative current was measured in the pH 7.0 phosphate buffer at 37 ℃. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode.
0 Figure 2.7 The detecting dynamic range of the
PVA-SbQ/Gox/PdS100-CPE biosensor. The glucose biosensor PVA-SbQ/GOx/PdS100-CPE was characterized for its detecting dynamic range by treated with different concentrations of glucose at 37 ºC in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode. Each data was obtained from three independent measurements.
(a) Figure 2.8 Reproducibility of the glucose biosensor PVA-SbQ/GOx/PdS100-CPE. The experiment was performed at 37 ℃ by repeatedly measuring the responses of glucose biosensor to 10 µM (a) and 1 mM glucose (b). The measurement was performed in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode. The relative standard deviations are 4.21%
and 8.62% for responses at 10 µM and 1 mM glucose, respectively.
0 50 100 150 200 250 300 350 400 450 500
0 1 2 3 4 21 25 28 50 88 96 101 108
Time (days)
Current (nA)
Figure 2.9 Long-term stability of PVA-SbQ/GOx/PdS100-CPE. The PVA-SbQ/GOx/PdS100-CPE was stored at 4 ºC before use. At the time points indicated in this experiment, the response of PVA-SbQ/GOx/PdS100-CPE to 100 µM glucose was determined at 37 ℃ in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode. Each data was obtained from three independent measurements.
Figure 2.10 Effect of pH on the response of PVA-SbQ/GOx/PdS100-CPE. The effect of pH to the response of PVA-SbQ/GOx/PdS100-CPE to 100 µM glucose was determined in phosphate buffer at 37 ℃ under different pH values (pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0). The working potential was set at 500 mV relative to the Ag/AgCl reference electrode. Each data is obtained from three independent measurements.
0 Figure 3.1 Responses of Glucose oxidase-immobilized NH2-Pd-CPE.
(a) Response of the glucose oxidase-immobilized CPE without NH2-plasma treatment. (b) Response of the glucose oxidase-immobilized NH2-CPE. The CPE was treated with 50W NH2-Plasma treatment prior to the immobilization of glucose oxidase. The glucose immobilization was carried out overnight at room temperature in the presence of 1 mg/mL glucose oxidase and 2% glutaraldehyde. The responses were determined at 25 ℃ in the presence of 10 mM glucose in a pH 7.0 phosphate buffer.
0 500 1000 1500 2000 2500
50W 100W 150W
Watt
Current(nA)
Figure 3.2 Effect of ionizing power on the treatment of Pd-CPE by
NH3 plasma. The available NH2- reactive sites on Pd-CPE surface can be affected by the power applied to the instrument during the plasma treatment. The NH2-reactive sites on Pd-CPE were indirectly indicated by the responses of covalently cross-linked glucose oxidase on the modified electrode. Three electordes were generated, X50-Pd-CPE, X100-Pd-CPE and X150-Pd-CPE, by treating with NH3 plasma under three different powers, 50W, 100W and 150 W, respectively. The responses of various GOx-X-Pd-CPEs was determined at 25 ℃ in a pH 7.0 phosphate buffer with 10 mM glucose. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode.
4000
0 500 1000 1500 2000 2500 3000 3500
0 100 200 300 400 500 600
T im e(sec)
Current(nA)
Figure 3.3 Successive response of GOx-X150-Pd-CPE. The successive responses of GOx-X150-Pd-CPE was determined at 25 ℃in a pH 7.0 phosphate buffer by step-wisely adding 500 µM glucose. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode.
0 Figure 3.4 Detecting dynamic range of GOx-X150-Pd-CPE. The
responses of GOx-X150-Pd-CPE electrode were determined with different concentrations of glucose at 25 ℃ in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode. Each data was obtained from three independent measurements.
0 Figure 3.5 Reproducibility of GOx-X150-Pd-CPE. The experiment was
performed at 25 ℃ by repeatedly measuring the responses of GOx-X150-Pd-CPE to 50 µM (a) and 500 µM glucose (b). The measurement was performed in a pH 7.0 phosphate buffer. The working potential was set at 500 mV relative to the Ag/AgCl reference electrode.
The relative standard deviations are 4.21% and 8.62% for responses at 10 µM and 1 mM glucose, respectively.
0 100 200 300 400 500 600 700 800 900 1000
0 1 2 3 5 6 7 9 10 12 26 35 41 49
Time(days)
Current (nA) 50 W
100 W 150 W
Figure 3.6 Long-term stability. The GOx/Pd electrode was treated with
1 mM glucose under the 500 mV potential in the phosphate buffer, pH 7, at 25℃.Each data was obtained from three measurements.
Appendix 1. The general configuration of a biosensor. The biosensor
is a devise having a biological sensing element either intimately connected to or integrated within a transducer. The aim is to produce a digital electronic signal, which is proportional to the concentration of specific chemicals. The directions of arrows indicate the process of measuring specific analyte.
Appendix 2. Conventional immobilization procedures. A numbers of techniques have been used to immobile biological molecules in carrier materials. The aim is to provide a compatible environment for biological element in order to produce rapid electron transfer at the electrode surface and store for a long term.
Appendix 3. Plasma modification procedures. Carboxylic acid groups
or amine functions, directly attached to the conducting support can be used for the covalent coupling of complementary amino groups of aspartic/glutaric acid residues, respectively.
Appendix 4. Conducting mechanism of conducting polymer. The
product of the reduction is a radical anion, with the intergap energy states occupied by the two electrons from one π-bond and the electron added by reduction. This state is known as polaron. Addition of a second electron to the same site yields a dianion, called a bipolaron.
Appendix 5 Synthesis of Polypyrrole. Polypyrrole is formed by
electrochemical oxidation of pyrrole at the anode and generates radical cations. Because the polymer is an electronic conductor of electricity, the deposition of polymer does not bring the process to a halt. Instead, polymer continues to be deposited until a relatively thick, freestanding film is formed.
Appendix 6. Interaction of ions with surface. When an ion approaches
the surface of a solid, one or all of the following phenomena may occur, such as reflected ions and neutrals, secondary electron, ion implantation, rearrangement and sputtering.
Appendix 7. Formation of a thin film. The sputtering atom will join
other single atom to form a group of atom ,called nucleation. These nuclear lead to the formation of island each containing high density of atom. Eventually they grow large enough to touch and then coalescence until the reaches continuity.
(a)
(b)
Appendix 8. Response of a reversible redox couple during a single
potential cycle. (a) Cyclic voltammetry consists of scanning linearly the potential of a stationary working electrode. Depending on the information sought, single or multiple cycles can be used. (b) A negative-going potential scan is chosen for the first half-cycle. As the applied potential approaches the E0 for the redox process, a cathodic current begins to increase, until a peak is reached. During the reverse scan, R molecules are reoxidized back to O and an anodic peak results.
Appendix 9. Concentration of oxidized and reduced form.
Concentration distribution of the oxidized and reduced forms of the redox couple at different times during a cyclic voltammetric experiment corresponding to the initial potential (a), to the formal potential of the couple during the forward and reversed scans (b,d), and to the achievement of a zero reactant surface concentration (c).
Appendix 10. The three-electrode system. (a) The character of working
electrode depends on what kind of reaction on itself. When oxidative reaction occurs, the working electrode is anode. When reductive reaction occurs, the working electrode is cathode. (b) Counter electrode takes place opposite reaction to working electrode. Counter could not influence the working electrode; it is usually made of platinum. (c) The main function of reference electrode is to set up the potential on the working electrode correctly.
Appendix 11. Schematic diagram of the batch operation of the amperometric sensor. Thermostat keeps the electrochemical cell under the desired temperature to avoid the unstable response from enzyme electrode. Personal computer will collect the electronic signal from working electrode and analyte these data.
Appendix 12. Schematic diagram of cross-link. Chemical modification of electrode could generate activated site for covalently bonding.
Cross-linking the enzyme layer associated with glucose oxidase with glutaric dialdehyde generate a stable, integrated enzyme electrode.
Appendix 13. Flow diagram of an IC fabrication process. A thin layer, such as an insulating silicon dioxide film, is deposited on a substrate. A light-sensitive photoresist layer is then deposited on top and patterned using photolithography. Finally, the pattern is transferred from the photoresist layer to the silicon dioxide layer by an etching process.