Home Search Collections Journals About Contact us My IOPscience Detection of cadmium sulphide nanoparticles by using screen-printed electrodes and a handheld device This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2007 Nanotechnology 18 035502 (http://iopscience.iop.org/0957-4484/18/3/035502) View the table of contents for this issue, or go to the journal homepage for more Download details: IP Address: 200.136.225.151 The article was downloaded on 26/11/2010 at 16:22 Please note that terms and conditions apply. INSTITUTE OF PHYSICS PUBLISHING NANOTECHNOLOGY Nanotechnology 18 (2007) 035502 (6pp) doi:10.1088/0957-4484/18/3/035502 Detection of cadmium sulphide nanoparticles by using screen-printed electrodes and a handheld device Arben Merkoçi1 , Luiz Humberto Marcolino-Junior2 , Sergio Marı́n1,3 , Orlando Fatibello-Filho2 and Salvador Alegret3 1 Institut Català de Nanotecnologia, Campus UAB, 08193 Bellaterra, Barcelona, Catalonia, Spain 2 Laboratório de Bioanalı́tica, Departamento de Quı́mica, Universidade Federal de São Carlos, Rod. Washington Luiz, km 235, CP 676, 13560-970-São Carlos/SP, Brazil 3 Grup de Sensors and Biosensors, Departament de Quı́mica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain E-mail: arben.merkoci.icn@uab.cat Received 21 September 2006, in final form 13 November 2006 Published 3 January 2007 Online at stacks.iop.org/Nano/18/035502 Abstract A simple method based on screen-printed electrodes and a handheld potentiostatic device is reported for the detection of water soluble CdS quantum dots modified with glutathione. The detection method is based on the stripping of electrochemically reduced cadmium at pH 7.0 by using square wave voltammetry. Various parameters that affect the sensitivity of the method are optimized. QD suspension volumes of 20 µl and a number of around 2 × 1011 CdS quantum dots have been able to be detected. The proposed method should be of special interest for bioanalytical assays, where CdS quantum dots can be used as electrochemical tracers. S Supplementary data files are available from stacks.iop.org/Nano/18/035502 (Some figures in this article are in colour only in the electronic version) as small fluorescent molecules like dyes etc. It is well known, despite the high detection limits and well known assay protocols, that enzymes suffer from stability problems and high cost as well as difficulties or near impossibility to carry out simultaneous detections. Fluorescent dye labels also are expensive, photobleach rapidly, and the equipment required to project the image of the dyes on an array surface is expensive and unwieldy [5]. In principle nanoparticles provide a novel platform for improving specific activity of a label as well as affinity to the tracer molecules (DNA probes or other biomolecules) [6, 7]. Nanosized particles have a chemical behaviour similar to small molecules and can be used as specific labels for DNA strands or antibodies. Nanoparticles in general and quantum dots [8] (QDs) in particular may be expected to be superior in several ways. Compared to existing labels, nanoparticles in general and QDs especially are more stable and cheaper. They allow more flexibility, faster binding kinetics (similar to those in 1. Introduction Inorganic compounds with nanometre dimensions—nanoparticles or quantum dots—are important because of their photovoltaic, photoelectrochemical, and electroluminescent applications including sensors and biosensors [1–4]. Recent advances in nanobiotechnology, and particularly the development of functionalized nanoparticles combined with advances in molecular biology research, provide the impulsion for the present explosion in DNA sensors and immunosensors among other fields. These devices are playing a growing role in various analytical applications where an accurate, low cost, fast and on-line measuring system is required. To improve the assay sensitivity and to achieve a better and more reliable analysis there is a great demand for labels with higher specific activity. The most used labels for DNA and immunosensors to date have been enzymes as well 0957-4484/07/035502+06$30.00 1 © 2007 IOP Publishing Ltd Printed in the UK Nanotechnology 18 (2007) 035502 A Merkoçi et al a homogeneous solution), high sensitivity and high reaction rates for many types of multiplexed assays, ranging from immunoassays to DNA analysis. Electrical methods for the detection of nanoparticles have been extensively used. Park et al [9] reported a conductivitybased DNA detection method utilizing oligonucleotidefunctionalized Au nanoparticles that provides an alternative to existing detection methods (see [6, 11] in the mentioned paper) and presents a straightforward approach to high-sensitivity and selectivity, multiplexed detection of DNA. Although conventional optical probes based on the fluorescence of nanoparticles have been successfully used, those based on electrochemical properties are still under research. The importance of electrochemical methods in nanoparticle based bioassays is extensively revised in the last review of Chem. Rev., dedicated to Nanostructures in Biodiagnostics [10] where it is emphasized among other things that ‘Electrical detection methods offer the possibility of portable assays that could be used in a variety of point-of-care environments’. The use of quantum dots as electrochemical labels for DNA and immunosensing has several advantages. The related technique—stripping voltammetry—compared to optical ones is cheaper, faster and easy to use in field analysis. Moreover, it offers the possibility for simultaneous detection of several biological molecules in the same sample and using a unique sensor owing to the distinctive voltammetric wave produced by different electrochemical tracers. An electrical immunoassay coding protocol for the simultaneous measurements of multiple proteins based on the use of different inorganic nanocrystal tracers have been developed. The concept is demonstrated for a simultaneous immunoassay of β 2-microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively [11]. The designed assay based on the nanocrystal dissolving shows the efficient coupling of the multiprotein electrical detection to the amplification feature of electrochemical stripping transduction yielding fmol detection limits. The offered advantages along with the possibility to be used in several biosensing systems based on electrochemical techniques require the development of novel nanoparticle detection strategies. Several works on DNA or immunoanalysis based on gold nanoparticle [12–18] or quantum dot [19] detection by using stripping techniques have been reported. The majority of these electrochemical methods have used chemical dissolutions of gold nanoparticles (in a hydrobromic acid/bromine mixture) or quantum dots (with nitric acid) followed by accumulation and stripping analysis of the resulting metal ion solutions. The HBr/Br2 solution is highly toxic and therefore methods based on direct electrochemical detection of gold nanoparticle tags have been developed [20, 21] and even applied for DNA analysis [22]. Regarding the direct electrochemical detection of quantum dots so as to achieve a full integration of DNA electrochemical sensors there is still much work to be done. The following work shows a simple method for the direct detection of cadmium sulphide quantum dots in neutral solution medium (pH ∼ 7.0) without the need for chemical dissolution. It is based on dropping a few microlitres of CdS QD suspension on the surface of a screen-printed electrode and Figure 1. Upper part: image of the hand held device system used for CdS QD detection. The principal components are the potentiostat, the palmtop PC and the screen-printed electrodes (SPE). Lower part: schematic diagram of the direct voltammetric detection of the CdS QDs using an SPE. the subsequent square wave voltammetry detection based on the reduced cadmium formation and stripping, giving a well shaped and sensitive analytical signal. The proposed method can be easily extended for other quantum dots based on other heavy metals, offering new opportunities for applications in electrochemical DNA genosensors. 2. Experimental details 2.1. Apparatus All voltammetric experiments were performed using a PalmSens (Palm Instrument BV, Houten, The Netherlands) that consists of a portable potentiostat interfaced with a palmtop PC (155 mm × 85 mm × 35 mm) (see figure 1, upper part). Electrochemical experiments were carried out using a screen-printed electrode (SPE) (Palm Instrument BV, Houten, The Netherlands). The screen-printed electrochemical cell consists of a graphite working electrode (diameter 3 mm), a graphite counter-electrode and a silver pseudo-reference electrode. Electron microscopy (TEM) of CdS QDs was performed on a JEOL JEM-2010. 2.2. Reagents Synthesis of CdS QDs was carried out using Schlenk techniques under nitrogen. Chemicals and solvents were used as received from Sigma-Aldrich: cadmium perchlorate, hexamethyldisilathiane (HMSDT), and tetramethylammoniumhydroxide (TMAH). All solutions were prepared in doubly distilled water. Potassium dihydrogenphosphate, phosphoric acid, and sodium 2 Nanotechnology 18 (2007) 035502 A Merkoçi et al phosphate buffer solution, pH 7.0). After this, using the same sensor, a determined volume of the QD suspension is added and measurements (SWV) performed by using the ‘sample’ option. In this way the subtracted curve was obtained. No special activation of the electrode surface was used for further experiments with the same sensor (up to six or seven measurements). hydroxide were purchased from Sigma-Aldrich; hydrochloric acid (37% m/m) was purchased from PanReac (Barcelona, Spain). Appropriate dilutions from this stock solution were also prepared in 0.1 mol l−1 phosphate buffer solution (pH 7.0) prior to each set of measurements. 2.3. Preparation of CdS quantum dot suspension The preparation method is based on arrested precipitation of water dispersed cadmium with sulphide precursors [23]. According to this method 3.228 g glutathione as modifier and 0.799 g CdCl2 were first dissolved in 176 ml water and stirred during 5 min. Subsequently 8.5 ml TMAH (tetramethylammoniumhydroxide) and 315 ml ethanol were added and after 10 min the precursor solution was thoroughly degassed. 0.738 ml HMDST is added quickly onto the degassed precursor solution, giving a clear (slightly yellow) colloidal solution of glutathione-coated CdS nanoparticles. The mixture was magnetically stirred for 1 h and the prepared particles were precipitated by adding tetrahydrofuran (THF). One day later the supernatant was decanted and the precipitate was dissolved in water/THF mixture and precipitated again with THF to remove excessive reagents and reaction by-products, respectively. Finally, the supernatant liquid was decanted and the precipitate was dried under vacuum (<1 mbar). The powdery CdS nanoparticles were dissolved again in water, obtaining a clear colloidal solution. A stock solution of 1.86 × 1019 CdS QDs ml−1 in 0.1 mol l−1 phosphate buffer solution (pH 7.0) was prepared and used in further experiments. SWV of cadmium ion solution, before CdS QD detection, was previously studied by using the SPE. Two detection methods were performed. According to the first one the SPE was immersed into 20 ml of a 0.1 mol l−1 HCl solution and the SWV performed following the classical procedure: accumulation of cadmium ions under stirring conditions and than stripping in a quiescent solution. The results obtained (not shown) were similar to those reported by Palchetti et al [24]. Besides this method we pushed the research to the detection of small sample volume so as to achieve later on detection of lower QDs solution volumes. According to this second method a volume as low as 20 µl of cadmium solution dropped onto the SPE surface has been able to be detected. (Supporting information, figure 1S, available at stacks.iop.org/Nano/18/035502). A linear range of response from 10 to 1000 ppb with a detection limit of around 5 ppb cadmium ion was obtained. 2.4. Electrochemical detection of CdS QDs 3.2. Detection of CdS QDs The measurements were performed suspending a volume of 20 µl under the sensor stripped in the horizontal position, to ensure electrical contact (complete circuit). Each SPE was pretreated, before using, by applying −1.1 V for 300 s, and then square wave voltammetric (SWV) scans were carried out until a low and stable background was obtained. SWV experiments were performed to evaluate the electrochemical behaviour of the screen-printed electrode (SPE) for CdS QD detection. The proposed protocol (figure 1, lower part) involves the introduction of CdS QDs on the surface of the SPE (figure 1(A)). During this step a drop of 20 µl containing an appropriate concentration of CdS QDs (ranging from 0.5 to 14.0 × 1016 QDs ml−1 ) was suspended onto the SPE for 60 s and a potential of 0 V was applied. The second step was the accumulation step. In this step (figure 1(B)) a deposition potential of −1.1 V for 120 s was applied to promote the electrochemical reduction of Cd2+ ions contained in the CdS QD structure to Cd0 . After the accumulation step, SWV was performed. During this step (figure 1(C)) the potential was scanned from −1.1 to −0.7 V (step potential 10 mV, modulation amplitude 30 mV and frequency 15 Hz), resulting in an analytical signal due to the oxidation of Cd0 . After the SWV measurement the SPE was manually cleaned (figure 1(D)) with a 0.1 mol l−1 phosphate buffer solution (pH 7.0). A blank subtraction method was performed. The blank was measured using a separate blank solution (0.1 mol l−1 The direct detection of CdS QD suspension on SPE represents a novel approach with special interest for electrochemical characterization of QDs and further applications in biosensing based on labelling. During the deposition time, applying a potential of −1.1 V, reduced cadmium, necessary for further stripping, could have been obtained via two mechanisms (see figure 2). 3. Results and discussion 3.1. Detection of cadmium ion solution dropped onto SPE surface First mechanism. The cadmium ions in equilibrium with CdS nanoparticles can be directly reduced according to (CdS)Cd2+ + 2e → (CdS)Cd0 . (1) Second mechanism. The direct reduction of CdS nanoparticles: CdS + 2H+ + 2e → Cd0 + H2 S (2) in analogy with the cathodic reduction of PbS nanoparticles in water solution [25], that depends on solution pH. (This is also confirmed by the pH effect studies. See the following sections.) The CdS QDs are reduced in this way to Cd0 while applying a potential of −1.1 V for 120 s. After this ‘preconcentration’ the redissolution of the Cd0 formed occurs, giving the SWV response that depends on the number of CdS QDs. To get further insight into the response mechanism of CdS QD detection linear sweep voltammetry (LSV) was also performed. The plot of the corresponding peak current versus the square root of the scan rate (Supporting information 3 Nanotechnology 18 (2007) 035502 A Merkoçi et al Figure 2. Schematic diagram of the CdS nanoparticle detection mechanisms either through reduced cadmium formation in acidic (left) or neutral (right) medium. figure 2S, available at stacks.iop.org/Nano/18/035502) did not show a reversible system with diffusion control of the cadmium redox reaction. Instead a mixed phenomenon (probably coming from QDs adsorption) could have occurred, which can be supported by the obtained plot curvature. This is due also to the rate of mass transport (diffusion) for CdS QD nanoparticle redox systems, which seems to be slower than that of the cadmium ions, similar to that for Fe2 O3 nanoparticles [26]. The effect of pH on the SWV of CdS QD suspension was studied. The results obtained (figure 3(A)) show that the peak current decreases on increasing the pH, being more significant from pH 7.0 to 3.0. This general augmentation of response is related to the increase of the reduced cadmium produced by an enhanced CdS reduction by decreasing the pH (according to equation (2)). The results obtained show that even at pH 7.0 the SWV signal was around half of that obtained at pH 3.0 and so sufficient for further analytical use. Moreover, the use of pH 7.0 is of interest, taking into consideration future applications in DNA sensing, being the usual medium pH in hybridization procedures. The SWV parameters were optimized so as to obtain the highest peak signal for CdS QD detection. Figures 3(B)– (E) show the optimization results for a 20 µl drop of QD suspension at pH 7.0 phosphate buffer. A maximum response at 25 Hz was obtained while changing the frequency from 15 to 35 Hz (figure 3(B)), being the modulation amplitude maximum at 30 mV at an operation range from 10 to 40 mV (figure 3(C)). The effect of deposition potential upon QD detection was also studied. As expected, the current response was increased by decreasing the deposition potential from Figure 3. (A) Effect of pH; (B) SW frequency; (C) modulation amplitude; (D) deposition potential; (E) deposition time to the SPE response for CdS QDs (4.65 × 1017 mol l−1 ) in a solution of 0.1 mol l−1 phosphate buffer (pH 7.0). Square-wave voltammetric scan with frequency of 25 Hz, step potential 10 mV and amplitude of 30 mV. Deposition potential of −1.1 V during 120 s. Figure 4. Performance of ten different sensors during measurements with a 14 × 1016 QDs ml−1 concentration suspension. Others experimental conditions as in the figure 3. −1.0 to −1.2 V. An unexpected decrease was obtained on decreasing the potential to −1.3 V (see figure 3(D)). This QD current decrease is related to an inhibition of the response at 4 Nanotechnology 18 (2007) 035502 A Merkoçi et al Figure 5. Square-wave stripping voltammograms after blank subtraction for increasing concentration of QDs: (a) 0.5, (b) 2.0, (c) 4.7, (d) 7.4, (e) 9.3, (f) 12.1, (g) 14.0 × 1016 QDs ml−1 . Also shown is the corresponding calibration plot (right) over the range 0.5–14.0 × 1016 QDs ml−1 . The measuring solution was 0.1 mol l−1 phosphate buffer, pH 7.0. Square-wave voltammetric scan with frequency of 25 Hz, step potential 10 mV and amplitude of 30 mV. Deposition potential of −1.1 V for 120 s. more negative potential values, probably due to an opposite effect coming from glutathione. The increase of deposition time better supports the QD detection due to the increase of the quantity of the reduced cadmium coming from the cadmium ions in equilibrium with CdS QDs at the given conditions. The response of diverse SPEs to the same drop of a 14 × 1016 CdS QD concentration suspension was also studied (figure 4). The results obtained show an RSD of 6.73%. The stability of the response for the same SPE was also studied (Supporting information figure 2S, available at stacks.iop.org/Nano/18/035502). For this case, an RSD of 11.8% was obtained for up to six measurements and it was almost doubled (22.2%) for four consecutive measurements. These results show that better responses are obtained by using one SPE for each measurement. Figure 5 shows typical square wave stripping voltammograms after the blank subtraction for increasing concentration of CdS QDs up to 1016 QDs ml−1 . The corresponding calibration plot including the error bars for a set of three parallel measurements is also shown. CdS QDs as low as 1016 ml−1 have been possible to detect. Taking into consideration the CdS QD drop volume introduced (20 µl), this corresponds to a detection limit of around 2 × 1014 CdS QDs. during which the electrochemical reduction of CdS QDs to Cd0 occurs. After the accumulation step SWV was performed by scanning from −1.1 to −0.7 V, resulting in an analytical signal due to the oxidation of Cd0 . The analytical signal used for the CdS QDs quantification comes from a mixed phenomenon detection which depends on the medium pH. The analytical protocols have been optimized to give results which display a wide, linear response range as well as a CdS QD detection limit that is of interest for various applications ranging from DNA analysis to immunoassays. The proposed technique represents a lower cost alternative to optical methods and will be of interest for fast screening as well as in field analysis. 4. Conclusions References Acknowledgments This work was financially supported by the Spanish ‘Ramón Areces’ foundation (project ‘Bionanosensores’) and MEC (Madrid) (projects MAT2005-03553, BIO2004-02776, Nanobiomed CONSOLIDER). The authors acknowledge the skilful experience of Mrs Anna Puig in all the technical support given to this work. Special acknowledgement to CAPES (Brazil) for financial support of LHM and OF. [1] [2] [3] [4] A direct detection technique for CdS QDs, that can be extended for other similar QDs, that avoids the chemical dissolving as in the previously reported electrochemical detection methods is achieved. The hypothesis for the detection is explained in the light of the experimental results obtained. 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