КОЛЛОИДНЫЙ ЖУРНАЛ, 2007, том 69, № 5, с. 703-708
УДК 541.183.22:547.97+541.13
IMMOBILIZATION OF PRUSSIAN BLUE NANOPARTICLES ONTO THIOL SAM MODIFIED Au ELECTRODES FOR ANALYSIS OF DL-HOMOCYSTEINE
© 2007 Jianrong Chen, Yuqing Miao, Xiaohua Wu
Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces Zhejiang Normal University
Jinhua 321004, China Поступила в редакцию 20.11.2006 г.
Prussian blue (PB) nanoparticles were immobilized onto gold electrodes using L-cysteine, 1,3-propanedithiol and 1,8-octanedithiol as a bridge between the gold surface and the PB nanoparticles by the self-assembly method. The obtained PB/thiol/Au electrodes exhibit direct and indirect electrocatalytic activity toward DL-ho-mocysteine (HCys) oxidation. It is possible for these PB nanoparticles modified electrodes to be used for the determination of HCys.
1. INTRODUCTION
Thiols are a class of compounds characterized by the presence of sulfhydryl residues. The biological thiol, DL-homocysteine (HCys), is of practical present interest. It exhibits biological importance in various metabolic pathways, and also has numerous roles in many metabolic diseases [1]. In the last decade HCys has also been proposed as an independent risk factor in the development of premature occlusive vascular diseases [2]. Hence, the accurate and quick detection and quantifying of HCys are of extremely important [3].
However, HCys measurement has always proven to be difficult. Most of the methods suffer from increased cost and complication of analysis due to the need of de-rivatization. Compared to spectrophotometric methods, electroanalysis is a more attractive option because it has the advantages of simplicity and high sensitivity. However, the direct oxidation of thiols at conventional electrodes is slow and requires a high potential. Therefore, various novel materials or chemically modified electrodes have been employed for amperometric detection of thiols at low applied potentials. The study of Chailapakul [4] shows that boron-doped diamond electrodes have better catalytical sensitivity toward the electrooxidation of sulfur-containing compounds. The use of a carbon-nanotube paste electrode also provides an effective means for the determination of homocys-teine [5]. A decrease of ca. 120 mV in the overpotential for the oxidation of homocysteine compared to a traditional carbon paste electrode, is reported along with greatly enhanced signal-to-noise characteristics. Similarly, the carbon-nanotube modified electrodes for electrocatalytic oxidation of thiols have been reported by Salimi et al. [6].
Recently, Prussian Blue (PB) has been demonstrated to allow the detection of some important thiol at low
applied potential. The electrocatalytic behavior of Prussian blue towards thiol oxidation was thoroughly investigated by Ricci et al. [7, 8]. PB has been known for its catalytic activity towards H2O2, and it is used often to detect H2O2 or to fabricate oxidase-based biosensors. Only extremely limited reports have been involved in the use of PB as catalysis for the electrochemical detection of thiols.
In this paper, we have employed L-cysteine (Cys), 1,3-propanedithiol (PDT) and 1,8-octanedithiol (ODT) to modify the gold surface. The purpose is to anchor PB nanoparticles and develop electroanalytical method for the detection of HCys.
2. MATERIALS AND METHODS 2.1. Materials
Cys, PDT, ODT and HCys were purchased from BBI, Shanghai Dere Finechem, Acros Organics and Fluka, respectively. They were used as received. All other chemicals were of analytical grade and were used without further purification. All solutions were prepared by ultra-pure water from Milli-Q water. All experiments were carried out at room temperature (approximately 20°C).
2.2. Preparation of PB nanoparticles
PB nanoparticles were prepared by mixing equimo-lar amount of FeCl3 and K4[Fe(CN)6] aqueous solutions. 2 ml K4[Fe(CN)6] (2 mM) was mixed with 1ml water solution of 0.1 M KCl and 0.01 M HCl to constitute solution I. 2 ml FeCl3 (2 mM) solution was slowly dropped into solution I under vigorous stirring. A blue solution was gradually formed. The vigorous stirring was continued overnight to make the reaction com-
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Fig. 1. TEM images of PB nanoparticles.
plete. The obtained colloid solution was stable during one week.
2.3. Preparation of Self-Assembled Monolayers (SAMs) of thiols
Piranha solution is used to remove organic residues from substrates. The gold electrodes (2 mm in diameter) were soaked in a 3 : 1 mixture of sulfuric acid and 30% hydrogen peroxide for 20 min followed by a thorough rinse with ultra-pure water and ethanol. After that, the electrode was scanned between - 0.1 and 1.2 V until a steady-state current-voltage curve was obtained in 0.1 M KCl.
2 mM Cys was prepared in ultra-pure water, and 2 mM PDT or ODT was done in ethanol. SAMs were formed by immersing the freshly cleaned gold electrodes in Cys, PDT or ODT solution overnight. Upon removal from the deposition solution, the substrate was thoroughly rinsed with ethanol and water to remove the physically adsorbed species. The resulting SAM modified electrode was dried in air and are defined as Cys/Au, PDT/Au and ODT/Au respectively.
2.4. Immobilization PB nanoparticles onto thiol modified Au
5 pi PB solution was cast onto the surface of thiol modified Au electrode. Then a centrifuge microtube was capped on the electrode to prevent PB colloid solution from drying. The process lasted overnight at 4° C. The formed PB/thiol/Au electrodes were rinsed with ultra-pure water.
2.5. Apparatus and Methods
Cyclic voltammetry experiments were performed with an Autolab PGSTA30 electrochemical instrument.
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Fig. 2. Cyclic voltammogramms (CVs) of (1) Cys/Au and (2) PB/Cys/Au in 0.1 M KCl electrolyte at the scan rates of 100 mV/s. The inlet is the CV of a PB/Cys/Au electrode between -0.1 and 1.0 V.
Gold electrodes with or without modification were used as working electrodes. Pt wire and saturated calomel electrode (SCE) were used as counter electrode and reference electrode, respectively. The potential scan rate in cyclic voltammetry experiments was 100 mV/s. The supporting electrolyte was 0.1 M KCl solutions, unless stated otherwise.
Transmission electron microscopy (TEM, JEM-100C-II) was used for the physical characteristics of the PB nanoparticles. The sample was prepared by placing an aqueous drop of PB onto a copper grid.
3. RESULTS AND DISCUSSION
Mixing of stoichiometric amounts of K4Fe(CN)6 and FeCl3 leads to the formation of PB nanoparticles. Pre-experiment shows the adding of 1 ml water solution with 0.1 M KCl and 0.01 M HCl during preparation of PB is necessary to obtain well defined electrochemical properties of anchored PB nanoparticles. Figure 1 is the TEM images of PB nanoparticles. As Fig. 1 shows, the formed PB nanoparticles are dispersed well. The size of PB nanoparticles is homogenous and the diameter is about 10 nm. The surfactant-free PB nanoparticles are stable during one week.
Cyclic voltammetry is a valuable tool for probing the nature of modified electrodes. Figure 2 shows the CVs of the Cys/Au and PB/Cys/Au in 0.1 M KCl solu-
0
0
tion at a scan rate of 100 mV/s. The low current response is observed for Cys/Au due to the hindrance of Cys SAM (Fig. 2, curve 1). It has been know that Cys can adsorb spontaneously through the strong sulfur-gold interaction onto the metallic surfaces such as Au, Ag, Pt, etc. It forms a highly ordered surface with few defects and exhibits a high degree of orientation, molecular ordering and packing density. Curve 2 in Fig. 2 shows that when PB nanoparticles were attached to the Cys/Au electrode surface, a couple of well-defined redox waves are observed with a peak-to-peak separation of about 46 mV at 100 mV/s. The couple of redox peaks between 0 and 0.4 V is attributed to Prussian white (PW) to PB conversion. This demonstrates that PB nanoparticles have been successfully assembled on Au surface and provide the necessary conduction pathways. Here, PB nanoparticles are immobilized by electrostatic interaction between negatively charged PB and positively charged Cys. Since isoelectric point of Cys is 5.02, it is positively charged in HCl solution. The cysteine is a bridge between the gold electrode and the nanoparticles, so it is essential for the formation of adherent nanoparticles. No characteristics redox peaks are observed when the working electrode was prepared by dropping PB colloid solution onto the surface of Au without Cys modification. It means that it is impossible for PB to be anchored at bare Au surface. However, the redox waves of PB between -0.1 and 0.4 V have a less symmetric nature. Also, a shoulder peak of reduction is shown at about 0.152 V. The similar phenomenon was mentioned by Liu et al. [9]. They reported a different method for preparation of PB nanoparticles. Those PB nanoparticles were also immobilized at Cys modified electrodes. They tried to explain the shoulder peak by the difference of nature between the bulk PB and the surface atoms on the nanoparticles. However, it seems advisable to attribute the observed current spike and shoulder peak of reduction process to the uncoordinated Fe(CN)6 [10]. As seen from the inset of Fig. 2, two pairs of peaks appeared between -0.1 and 1.0 V corresponding to the characteristic redox waves of PB. The results further confirmed that PB was assembled onto modified surface and still kept its electrochemical properties.
The dependence of the peak current on the scan rate has been studied for the PB/Cys/Au electrodes. As shown in Fig. 3, the peak potentials widen with the rising of scan rate, while the peak currents increase. The anodic and cathodic peak currents are both linearly proportional to the scan rat
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