2.1 Materials
Tetrachloroauric (III) acid trihydrate (HAuCl4･3H2O), cysteamine hydrochloride,
4-aminophenylacetic acid (CMA), hydrogen peroxide solution (30%), potassium
hexacyanoferrate (III) (approx. 99%), potassium dihydrogenphosphate, bovine serum albumin
(BSA) and N-(3-dimethylaminopropyl)-N’-ethylcarbodimide hydrochloride were purchased
from sigma. Glucose oxidase from Aspergillus niger was obtained from Sigma. Sodium
citrate, sodium nitrate, hydrogen chloride, glucose, sodium chloride (99.5%), potassium
chloride (99.5%), disodium hydrogenphosphate (99.0%), sodium dihydrogenphosphate
anhydrous, N-hydroxysuccinimide and 2-[N-morpholino] ethanesulfonic acid (99%) were
bought from Sigma and Showa.
2.2 Apparatus
A potentiostat CHI 440 (CH Instruments, West Lafayette, IN, USA) connected to a
personal computer was used for the measurement of electrochemical responses of biosensors
to hydrogen peroxide (H2O2). The three-electrode electrochemical system contained a
working electrode screen-printed carbon paste electrode (SPCE), a counter electrode (a
platinized electrode) and a reference electrode (Ag/AgCl). The oxygen plasma pre-treatment
of the surface of SPCE was performed on Diener electronic (type: Zepto/Atto).
2.3 Preparation of gold nanoparticles (AuNPs)
The preparation of gold nanoparticles was carried out by the conventional
Turkevich-Frens citrate reduction method [57]. The double-deionized H2O (200 mL) in a 500
mL Erlenmeyer flask was first heated to boiling. After boiling, 0.2 mg HAuCl4 and 0.1 g
sodium citrate were added into the flask with gently shacking to allow the chemicals to
dissolve. Seal the bottle by a piece of aluminum foil to prevent water evaporation and allow
the reaction to proceed until the appearance of the wine-red color (about 10 min). Stop the
reaction by putting Erlenmeyer flask on ice for about 10 min. The synthesized gold
nanoparticles were characteristics by determining absorption peak and the absorbance on the
U-3010 spectrophotometer (Hitachi, Japan). The approximate size and the concentration of
the synthesized gold nanoparticles can be estimated by the Beer’s Law. The AuNPs solution
was then stored in an aluminum foil-shield flask in dark at 4ºC.
2.4 Preparation of cysteamine-modified AuNPs
The cysteamine (CNH2)-modified AuNPs (CNH2AuNPs) was prepared by incubating
AuNPs solution and CNH2 at the final concentration of 10 nM and 0.03 mM, respectively, at
room temperature for two hours [58]. The stock CNH2 solution was prepared by dissolving
CNH2 in d.d. H2O to a concentration of 20 mM. After incubation the CNH2AuNPs solution
was collected by centrifugation at 13,200 rpm and 4ºC for 20 min. After removing supernatant
the CNH2AuNPs pellet was suspended in pH 6.0 phosphate buffer saline (PBS) (137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4) containing 0.1% BSA, to prevent
the aggregation. The prepared CNH2AuNPs solution was then characterized by determining
the absorption peak and the absorbance at the peak [59]. The prepared CNH2AuNPs could be
stored at 4ºC or used directly.
2.5 Pretreatment and modification of screen-printed carbon paste electrode
Prior to the assembly of three-electrode electrochemical system, the screen-printed
carbon paste electrode (SPCE) was pretreated with oxygen plasma under the room
temperature at 25 W for 30 sec to increase the hydrophilicity and the electrochemical
properties of the SPCE by removing the surface organic pollutants [60]. Subsequently, the
electrode was then washed by cyclic voltammetry within the potential range from -1.0 to 1.0
V at a scan rate 100 mV/s [61], followed by immersing in pH 7.0, 1X PBS before the
modification. After washing a thin layer of 4-carboxymethyl aryl diazonium (CMA) was
electrodeposited on the SPCE. Briefly, 8 mL 4-aminophenylacetic acid in ethanol (30 mM)
was mixed with 1 mL HCl (20 mM) and 1 mL NaNO2 (20 mM) to give the final concentration
of 24 mM, 2 mM and 2 mM, respectively. Then the diazonium cation solution was obtained
by incubating above mixture in ice-cold water with stirring for 10 min to allow the amino
group of the 4-aminophenylacetic acid to be diazoted. In a diazoted 4-aminophenylacetic acid
solution (24 mM) the diazonium salt was deposited on the SPCE surface by cyclic
voltammetry with the scanning range of -0.7 to 0.8 V and a scan rate of 200 mV/s for 10
cycles. The CMA-modified SPCE was then washed with pH 7.0, 1X PBS, twice and stored at
4ºC [62].
2.6 Modification of CMA-coated SPCE with CNH2AuNPs
The surface of CMA-deposited SPCE (CMA/SPCE) was first activated by cross-linking
reagent EDC and NHS before the immobilization of CNH2AuNPs and GOx [63]. Briefly, the
CMA-deposited SPCE was immersed into 0.1 M MES, pH 6.0 buffer solution containing 0.04
mg/mL EDC and 0.06 mg/mL NHS at room temperature for 30 min [64]. After incubation, the
surface activated, CMA-deposited SPCE was rinsed once with d.d. H2O to remove excess
unreacted EDC/NHS. Subsequently, 5 µL CNH2AuNPs solution (0.58 nM) was dropped
directly onto the surface of CMA/SPCE. The cross-linking reaction was allowed to be
proceeded by placing at room temperature for 30 min first and then staying at 4ºC for
overnight to form CNH2AuNP-coated CMA/SPCE electrode (CNH2AuNP/CMA/SPCE).
2.7 Generation of glucose oxidase electrode
Two types of glucose biosensor was generated and characterized. The first glucose
sensor is to immobilize glucose oxidase (GOx) on the CNH2AuNP/CMA/SPCE
(GOx/CNH2AuNP/CMA/SPCE), forming a sandwich-like structure by cross-linking [65]. The
CNH2 moiety on CNH2AuNPs allows the immobilization of other biomolecules such as
enzymes, antibodies and other protein on the surface of AuNPs [66]. The second glucose
biosensor was to immobilize GOx and CNH2AuNPs mixture on the surface of activated
CMA/SPCE (GOx- CNH2AuNPs/CMA/SPCE). GOx can also interact with the activated
CMA/SPCE to generate GOx/CMA/SPCE, which was used as a control.
Prior to immobilizing on the CNH2AuNP/CMA/SPCE or CMA/SPCE, GOx was first
activated by mixing 10 µL GOx (184 Units/µL) with 10 µL PBS, pH 6.0 containing 0.04
mg/mL EDC and 0.06 mg/mL NHS under room temperature for 30 min [67]. After activation,
GOx (approximate 90 Units) was either mixed with CNH2AuNP (0.58 nM) in a ratio of 1:1
(v/v) and deposited on the CMA/SPCE [68] or directly deposited on the
CNH2AuNP/CMA/SPCE. The cross-linking reaction was allowed to proceed at room
temperature for 30 min, followed by incubating at 4ºC overnight to form covalent bonding.
Two types of GOx electrodes were constructed. The first is GOx- CNH2AuNPs/CMA/SPCE,
a glucose sensor with GOx and CNH2AuNPs mixture immobilizing on the CMA/SPCE. The
second on is GOx/CNH2AuNPs/CMA/SPCE, a glucose sensor with GOx immobilizing on the
CNH2AuNPs/CMA/SPCE.
2.8 Cyclic voltammetry and electrochemical measurements
Cyclic voltammetry (CV) was widely used to characterize the electrochemical properties
of electrodes. CV is a powerful method for qualitative or quantitative analysis of the
electrochemical reactions. The experiment was usually performed on the conventional
three-electrode electrochemical system. The working electrode is supplied with a linearly
changed potential to trigger the corresponding electrochemical responses or currents, which
would be subsequently recorded and analyzed [69].
Cyclic voltammetry was also adopted to clean the surface of electrode by the redox
reactions. The cyclic voltammetry for either electrode cleaning or electrochemical analysis of
H2O2 was performed in PBS, pH 7.0 with a scanning range between -1.0 and +1.0 V and a
scanning rate of 100 mV/s for 5 to 10 cycles. Current-time responses of H2O2 on
AuNPs-coated SPCE or glucose on glucose sensors were carried out in PBS, pH 7.0 at the
fixed voltage of +0.7 V, followed by monitoring the electric redox current signals of
electrodes. It is worth to know that the measurement of electrochemical response of reactant
should not be carried out until resting current reached steady state. Current-time responses can
be used to evaluate the changes on electrode surface [70]. In this thesis, we used cyclic
voltammetry and current-time response to evaluate the effect of different AuNPs-based
modifications on the electrochemical properties of SPCE.