Anal. Chem. 2003, 75, 4667-4671
Technical Notes
Encoded Beads for Electrochemical Identification Joseph Wang,* Guodong Liu, and Gustavo Rivas†
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
Encoded redox beads, based on the encapsulation of different quantum dots (QD) within polystyrene microspheres, have been developed for electrochemical identification. Encoded redox rods, prepared by sequential plating of different metal tracers into the pores of a host membrane, have also been designed. By incorporating different predetermined levels of multiple metal markers, such redox-encoded particles lead to a large number of recognizable voltammetric signatures and, hence, offer great promise for covert tagging of commercial products. The resulting voltammetric signatures correlate well with the predetermined loading ratio, indicating a reproducible encapsulation process. As desired for effective authenticity testing, QD-based “identification layers” were reproducibly cast and removed from packages of commercial products to display their distinct voltammetric profiles. Factors affecting the preparation of such identification layers were optimized. The widespread dissemination of counterfeit products (such as drugs, perfumes, cigarettes, foods, or wines) is an escalating criminal activity across the globe. Analytical chemistry can play a major role in tracking products along the supply chain and protecting the consumers. Various analytical schemes have been proposed for the identification of counterfeit products. These include nucleic acid protocols employing distinct DNA sequences,1 near-IR spectroscopy,2 or separation techniques such as gas chromatography3 or capillary electrophoresis.4 Particle-based nanomaterials have received considerable recent attention for biological optical coding5,6 but have rarely been used for authenticity testing. Analogous nanomaterial-based electrochemical identification codes, with distinct voltammetric signatures, have not been reported for either biological coding or product protection. Here we report on a promising route for designing encoded redox beads for covert tagging of commercial products. The * Corresponding author. E-mail:
[email protected]. † Permanent address: INFIQC, Departamento de Fı´sico Quı´mica, Universidad Nacional de Co´rdoba, 5000, Co´rdoba, Argentina. (1) (a) Popping B. J. Biotechnol. 2002, 98, 107. (b) November AG, http:// www.november.de. (2) Frasson-Scaffi, S. H.; Pasquini, C. Analyst 2001, 126, 2218. (3) Marengo, E.; Aceto, M.; Maurino, V. J. Chromatogr., A 2002, 943, 123. (4) Saaverda, L.; Garcia, A.; Barbas, C. J. Chromatogr., A 2000, 881, 391. (5) Han, M.; Gao, X.; Su, J.; Nie, S., Nat. Biotechnol. 2001, 19, 631. (6) Walton, I.; Norton, S.; Balasingham, A.; He, L.; Ovison, D.; Gupta, D.; Raju, P.; Natan, M. J.; Freeman, R. G. Anal. Chem. 2002, 74, 2240. 10.1021/ac034411k CCC: $25.00 Published on Web 07/19/2003
© 2003 American Chemical Society
inherent miniaturization and low-power demands of electrochemical devices satisfy many of the requirements of field-based authenticity testing. Miniaturized electrochemical analyzers have thus been developed for a wide range of clinical or environmental applications7-9 but not for the task of product identification. The encoded redox particles, described in this report, are based on embedding different predetermined levels of multiple metal markers. This is accomplished by encapsulating various proportions of different quantum dots (QD) within polystyrene (PS) beads or by depositing various metal tracers onto the pores of a template membrane, in a manner analogous to the optical beads and rods of Nie5 or Keating and Natan,10 respectively. The resulting beads lead to a large number of recognizable voltammetric signatures, whose peak intensities reflect the predetermined level of the corresponding nanocrystals or metal stripes. Such distinct voltammetric fingerprints distinguish the individual particles from each other. This route offers a large variability of possible combinations in connection to a judicious design of encoded “identification” beads. Similar to optical (multicolor/ emission intensities) coding,5 m potentials and n current intensities will generate (nm - 1) distinct codes. For example, 999 distinctive codes are possible with 3 metal markers and 10 loading levels. Realistically, such scheme could generate up to 15 000 usable codes in connection with 5 potentials and 6 current intensities. The new electrical authenticity test, illustrated in Figure 1, involved casting of a QD-based identification layer on a package of a commercial product (A), its rapid dissolution (B), and voltammetric stripping quantitation of its metal constituents (C). The resulting stripping voltammogram (D) consists of multiple baseline-resolved metal stripping peaks related to the embedded ZnS, PbS, CdS, InAs, and GaAs nanocrystals. The characterization and electrochemical application of the new multimetal QD-tagged encoded beads and their application for covert tagging of commercial products are reported in the following sections. EXPERIMENTAL SECTION Apparatus. Square-wave voltammetric measurements were performed with a µAutolab Type II electrochemical system (Eco (7) Wang, J. Trends Anal. Chem. 2002, 21, 226. (8) Wijayamardhana, C.; Wittstock, G.; Halsall, B.; Heineman, W. R. Electroanalysis 1999, 12, 640. (9) Unwin, P. Instrumentation and Electroanalytical Chemistry. In Encyclopedia of Electrochemistry; Bard, A. J., Stratmann, M., Eds.; Wiley-VCH: Weinheim, 2003; Vol. 3. (10) Keating, C. D.; Natan, M. J. Adv. Mater. 2003, 15, 451.
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Figure 1. Schematic illustration of the electrochemical bar-coding system based on polystyrene beads loaded with different quantum dots (A), along with the dissolution of the QD-based identification layer (B), and stripping voltammetric detection of the metal tracers (C, D).
Chemie), controlled by the GPES software. The detection was carried out in a 1.5-mL glass cell using a 2-mm-diameter glassycarbon disk or a 2 × 4 mm screen-printed carbon (Acheson ink) working electrode (coated with preplated or in situ plated mercury films, respectively), a Ag/AgCl reference electrode, and a platinum counter electrode. Reagents. All stock solutions were prepared using deionized and autoclaved water. Nitric acid, octadecanethiol (ODT), CdCl2, ZnS, HCl, ZnCl2, CdCl2, PbCl2, petroleum ether, n-heptane, propanol, chloroform, and ethanol were purchased from Sigma. Acetonitrile, acetone, N,N-dimethylformamide (DMF), and polystyrene (MW 550 000) and heavy-metal atomic absorption standard (AAS) solutions (1000 mg L-1) were purchased from Aldrich. Polystyrene beads (1.2-µm diameter) were purchased from Bangs Laboratories. Preparation of QDs. Cadmium sulfide, lead sulfide, and zinc sulfide nanoparticles were prepared based on a slightly modified literature protocol.11 Fifty-milliliter aliquots of 10-4 M aqueous CdCl2, ZnCl2, or PbCl2 solutions (pH 5.8) and 50 mL of a 5 mM ODT solution (prepared in petroleum ether) were mixed into a conical flask; the resulting biphasic mixture was shaken vigorously. Hydrogen sulfide (prepared by mixing ZnS and HCl) was then bubbled through the stirred mixture for 20 min, during which the solution became yellow (CdS), white (ZnS), or black (PbS); the stirring was then stopped, and the nanoparticles were collected in the petroleum ether organic phase. The QDs/petroleum ether solution was rotary evacuated leading to a colored powder (yellow, white, or black depending on the specific sulfide). This powder was washed with ethanol to remove the excess ODT molecules. Subsequently, the powders were dispersed in chloroform. Incorporation of QDs into Polystyrene Beads (PS-QDs). The incorporation of QDs into the polystyrene beads was performed based on combining and modifying several literature protocols.5,12,13 Prior to such encapsulation, the PS beads were (11) Kumar, A.; Mandale, A. B.; Sastry, M. Langmuir 2000, 16, 9299.
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dried under vacuum. The different QDs (dispersed in chloroform) were first mixed in different predetermined volume ratios; the resulting suspension was mixed with propanol (95.0% v/v propanol, 5.0% v/v QD in chloroform). Subsequently, the dried polystyrene beads were added to the above mixture. The encapsulation process proceeded by shaking the resulting mixture for 30 min at room temperature. The beads were then dried under vacuum. Heptane was added to the dried beads, and the mixture was stirred for 50 min at room temperature followed by solvent decantation. The washing process was repeated until discrete particles were obtained. Finally, the beads were dried under vacuum. Preparation of Bismuth-Indium-Galium Multimetal Nanorods. Multimetal (bismuth-indium-galium) nanorods were prepared based on a slightly modified literature protocol.10 Alumina membranes with 200-nm pores and annular support rings (Whatman, Clifton, NJ), coated with a 0.5-1.0-µm silver layer, were used to prepare nanorods. Electroplating was performing on a CHI 440, controlled by CHI 2.06 software (CH Instruments, Austin, TX). The membrane was placed on a glass slide, with the silver side up. Silver was deposited to further seal the membrane using a constant current of -5 mA for 20 min in the presence of 100 mg L-1 silver AAS solution. The membrane was turned over on the aluminum foil, and additional silver was then plated into the membrane at -0.5 mA for 20 min to fill its branched sections. The silver AAS solution was then replaced by the corresponding diluted metal tracer AAS solution (100 mg L-1). Galvanostatic deposition of bismuth, indium, and galium proceeded at -5 mA for 20, 40, and 10 min, respectively. The membrane was rinsed with Nanopure water until the potential exceeded -4 V (prior to the deposition of the next metal). Finally, the membrane was rinsed with distilled water, and the sliver film backing was dissolved with 30% nitric acid for 10 min. The alumina membrane (12) Hu, M.; Noda, S.; Okubo, T.; Yamaguchi, Y.; Komiyama, H. Appl. Surf. Sci. 2001, 181, 307. (13) Hong, S. C.; Rief, U.; Kristen, M. O. Macromol. Rapid Commun. 2001, 22, 1447.
was then rinsed with distilled water and was dissolved by placing it in a 3 M sodium hydroxide for 30 min. The resulting suspension was centrifuged at 3000 rpm to sediment the particles. This process was repeated three times to remove the residual salt. The prepared Bi-In-Ga nanorods were dispersed in 1 mL of distilled water. Analytical Protocol. The identification layer (on commercial packages) was prepared by “sandwiching” the QD between two PS layers. For this purpose, a 20-µL polystyrene solution (50 mg mL-1 in DMF) was cast on the selected package and was allowed to dry. Subsequently, a 10-µL suspension of QDs-modified polystyrene beads (containing the predetermined QD levels) in water (2 mg mL-1) was placed on the first PS layer; once this layer was dried, an additional 20-µL aliquot of the polystyrene solution was on top and was allowed to dry. The resulting identification layer had a circular shape with a diameter of ∼6 mm. The “identification” signal was obtained by removing the QDcontaining PS layer from the commercial package (by peeling it with a plastic tweezer) and dissolving it in 120 µL of a DMF/1 M HNO3 (2:1) mixture; after a 2-min mixing/dissolution, the entire supernatant was transferred into the electrochemical cell containing 1.0 mL of the acetate buffer supporting electrolyte solution (0.2 M, pH 5.2). Square-wave voltammetric stripping measurements of the dissolved QDs and of the dissolved identification layer were performed using mercury film electrodes preplated or in situ plated onto glassy-carbon and screen-printed carbon, respectively. Prior to its coating, the glassy-carbon electrode was polished with a 0.05-µm alumina slurry and sonicated in 50% v/v M nitric acid, acetone, and deionized water for periods of 5 min (in each case). The mercury film-coated glassy-carbon electrode was prepared by applying a potential of -1.10 V for 20 min in a 0.1 M HCl solution containing 100 mg L-1 mercury ion. The metal detection protocol involved a 1-min conditioning step at -0.1 V, followed by a 2-min deposition at -1.40 V. Mercury-coated screen-printed electrodes were prepared in situ using a 1-min conditioning at 0.6 V, followed by deposition at -1.4 V for a given time (usually 30 s). These experiments employed an acetate buffer electrolyte solution (0.20 M, pH 5.2), containing 10 mg L-1 mercury and the dissolved identification layer. The stripping step was performed by scanning the potential between -1.20 and -0.30 V, using the square-wave mode, with a potential step of 50 mV, a 20-mV amplitude, and a frequency of 25 Hz. Baseline correction of the resulting voltammograms was performed using the “moving average” mode of the GPES (Autolab) software. Final results were obtained following background subtraction. Solid-state chronopotentiometric measurements of the encoded redox nanorods were performed with a potentiometric stripping PSU20 system (Radiometer), controlled by a PC using the TAP2 software (Radiometer). A 20-µL aliquot of a 0.01 mg mL-1 BiIn-Ga nanorod-dispersed solution was added to 100 µL of acetate buffer (0.2 M, pH 5.2). The Bi-In-Ga nanorods were collected by a 30-min precipitation on the surface of the mercury-coated screen-prints (deposited 10 min at 0.1 M HCl solution containing 100 mg L-1 mercury(II)). Chronopotentiometric measurements of the “collected” Bi-In-Ga nanorods were performed using a 1-min preconditioning at -0.10 V followed by 1 s at -1.1 V.
Figure 2. Square-wave stripping voltammograms obtained before (A) and after (B) the incorporation of QDs into the polystyrene beads. The predetermined loading of QD, 2.0ZnS/5.0CdS/2.0PbS. Conditions: A 5-µL aliquot of the polystyrene bead suspension was dissolved first in 10 µL of acetonitrile followed by addition of 10 µL of 1 M HNO3; the resulting 25-µL aliquot was transferred to an electrochemical cell containing 1.0 mL of acetate buffer solution (0.2 M, pH 5.2). Working electrode, preplated coated mercury glassy-carbon electrode; accumulation, 2 min at -1.4 V; square-wave voltammetric parameters, step potential, 50 mV, amplitude, 20 mV; frequency, 25 Hz.
Subsequent measurement was carried out (after a 10-s rest period) using an anodic current of +1.0 µA. RESULTS AND DISCUSSION Here we explore the ability to control the proportions of different QD within PS beads for creating a library of voltammetric codes. As desired for reliable product identification, such QDimpregnated microspheres offer a large variability of possible combinations. Each microsphere thus yields a characteristic multipeak stripping voltammogram, whose current intensities reflect the predetermined loading of the corresponding nanocrystal. Such an electrochemical stripping procedure is particularly promising for detecting metal-based identification beads, as it provides simultaneous measurements of up to five or six metals down to the subnanomolar concentration level and is highly compatible with decentralized testing.14-16 The remarkable sensitivity of stripping analysis also facilitates the use of negligible amounts of the metal markers, hence minimizing concerns regarding their toxicity. These capabilities are demonstrated below in connection with three-metal (Zn, Cd, Pb)-QD-based tagged beads as well as Bi-In-Ga striped nanorods. QD Encapsulation and Voltammetric Signatures. The ability to control the proportions of different QD within PS spheres and, hence, tune the signal intensities has a profound effect upon the success of the new identification scheme. Accordingly, we examined the agreement between the initial loading (encapsulation) ratio and the ratio of the intensities of the resulting voltammetric-peaks. Figure 2 displays such assessment for encoded peaks containing three QD (ZnS, CdS, PbS) markers. It compares stripping voltammograms for the dissolved nanocrystals before (A) and after (B) their encapsulation into the polystyrene (14) Wang, J. Stripping Analysis; VCH Publishers: Deerfield Beach, 1985. (15) Wang, J.; Tian, B.; Lu, J.; Yarnitsky, C.; Olsen, K.; Bennet, W. Anal. Chim. Acta 1999, 385, 429. (16) Jagner, D. Trends Anal. Chem. 1983, 2, 53.
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Figure 3. Reproducibility study of the electrochemical response obtained after dissolution of the QD-incorporated polystyrene beads. The inset shows stripping voltammograms for individual portions of the bead suspension. The predetermined loading of QD, 2.0ZnS/6.0CdS/2.0PbS. Other conditions, as Figure 2.
beads at a predetermined level 2ZnS/5CdS/2PbS. Well-defined voltammograms, with favorable and baseline-resolved zinc, cadmium, and lead peaks, are observed in both cases. Intensity levels of 2.0Zn/5.0Cd/2.3Pb and 2.0Zn/5.1Cd/2.1Pb are obtained before and after the incorporation, respectively. Such peak ratios indicate that the signals follow relatively closely the predetermined levels of the corresponding nanocrystals and that the loading ratio of the redox markers can be controlled. The low background and absence of additional redox signals indicate that the dissolution of the PS beads and the acetonitrile/nitric acid dissolution solution have no effect upon the voltammetric readout. The attainment of such good correlation between the predetermined loading ratio and the resulting stripping voltammogram, and of a high identification accuracy, requires a uniform and reproducible encapsulation process. The uniformity and precision of the “loading” process were examined by plotting histograms for each current intensity of dissolved triple-marker beads with a predetermined loading 2ZnS/6CdS/2PbS. Stripping voltammograms for six different aliquots of a suspension of the QD-loaded polystyrene beads are displayed in Figure 3, along with histograms for the corresponding peak currents. The resulting voltammograms are highly reproducible, reflecting the reproducibility of the encapsulation process; relative standard deviations (RSD) of 9.5, 6.8, and 3.4% were estimated for the corresponding zinc, cadmium, and lead peaks, respectively. The ratio of the peak intensities is 2.0Zn/7.1Cd/1.9Pb. It should be pointed out that possible aggregation of QD within the beads (that may affect their optical properties in analogous optical coding)5 is not a concern for the present electrical identification. Preparation and Detection of the Identification Layer. We evaluated different avenues for casting the QD-based identification layer on different surfaces (relevant to packages of commercial products). A sandwich PS/QD/PS preparation route (described in the Experimental Section) was found to be most compatible with a variety of surfaces (including paper, plastic, metal, or glass) and addresses concerns regarding the toxicity and leak of the QD markers. Such concerns are further minimized by encapsulating the QD within the PS beads. Such sandwich configuration had a circular shape (with a diameter of ∼6 mm), was durable over prolonged periods, was stable under harsh conditions, and offered convenient tagging of different surfaces. For example, the voltammetric profiles were not affected by heating the coated surface for 6 h at 50 °C or following a 2-week storage at room 4670 Analytical Chemistry, Vol. 75, No. 17, September 1, 2003
Figure 4. Square-wave stripping voltammograms obtained after dissolution of the identification layer immobilized in a commercial box. The voltammograms correspond to five different samples (a-e). The corresponding histograms are also shown as an inset. Conditions. Supporting electrolyte, 0.20 M acetate buffer solution pH 5.2. Working electrode, in situ plated mercury-coated screen-printed electrode. Deposition time, 30 s. Other conditions, as for Figure 2.
temperature. Different organic solvents were examined for dissolving the identification layer, including acetone, acetonitrile, chloroform, toluene, and DMF; the latter was found to be most effective. Combining DMF with nitric acid offered a fast (2 min) dissolution of the identification layer and its QD markers. The realization of effective authenticity testing requires a highly reproducible casting and dissolution of the identification layer. Figure 4 assesses the reproducibility of the casting and dissolution processes. It displays stripping voltammograms obtained after removing five different triple-marker identification layers (with a 2ZnS/5CdS/2PbS loading) from the cover of a commercial package (a-e). Well-defined voltammograms, with a flat baseline, and three resolved metal marker peaks are observed following a very short (30 s) deposition time. The stripping signals are reproducible, with RSD of 11.1, 2.2, and 10.3% for Zn, Cd, and Pb, respectively. The average signal intensity levels follow quite closely the predetermined QD loading (2.0ZnS/4.1CdS/2.0PbS vs 2.0ZnS/5.0CdS/2.0PbS); the
cadmium peak is somewhat smaller than the expected one. Overall, the data of Figure 4 indicate that the preparation of the identification layer is reproducible and that its dissolution step and HNO3/DMF dissolution medium have little effect upon the electrochemical detection and the identification process. A control experiment involving the removal of a QD-free PS layer led to a flat background, with no stripping peaks (not shown). This indicates that the dissolution and layer-removal (peeling) steps do not contribute to metal impurities that may affect the response. The fast (∼1 min) stripping-voltammetric detection and the use of single-use strip electrodes should facilitate practical identification applications. The speed of preparing the identification layer and its uniformity can be greatly improved in connection with modern microfabrication (microspotting) techniques. Redox-Encoded Nanorods. Another promising avenue for creating a library of electrical codes involves striped metal nanorods analogous to the optical metallic bar codes of Keating and Natan.10 Such preparation of redox-encoded nanorods relies on the sequential deposition of different metals onto the pores of a template membrane. This allows convenient and reproducible incorporation of a wide range of metal tags with favorable electrochemical properties and significantly lower toxicity (compared to QD-based redox beads). The resulting nanorods can be detected directly in connection to solid-state chronopotentiometry (through their direct contact with the transducer surface). Such elimination of the dissolution step greatly simplifies the measurement of identification redox particles. Each bar-coded rod thus yields a characteristic multimetal stripping potentiogram, whose potentials and intensities reflect the corresponding metals and the lengths of their stripe. For example, Figure 5 shows a characteristic chronopotentiogram for nanorods containing different segments of Bi, In, and Ga at a predetermined level 1Ga/4In/2Bi [prepared with 10 (Ga), 40 (In), and 20 (Bi) min depositions]. Well-defined and baseline-resolved peaks are observed, with the ratio of the peak intensities (1.00Ga/4.02In/1.96Bi) following very closely the predetermined metal loading. Such agreement reflects the reproducibility of the electrodeposition process. The baselineresolved peaks indicate that the number of distinguishable metals can be readily increased. CONCLUSIONS We have described the creation of a library of electrical codes based on encapsulating different predetermined levels of multiple
Figure 5. Solid-state chronopotentiograms of Bi-In-Ga multimetal nanorods with a predetermined stripe length ratio of 2:4:1. Precondition, 1 min at -0.1 V and 1 s at -1.1 V; measurement using a constant current of 1 µA.
semiconductor nanocrystals into polystyrene carrier beads or depositing various metal tracers onto the pores of a host membrane. The resulting electrochemical signatures correlate well with the predetermined loading ratio and indicate a reproducible encapsulation process. Identification layers, based on such redoxencoded beads, can be reproducibly cast (onto commercial packages) and dissolved to yield distinct voltammetric profiles. As desired for electrochemical identification, the number of electrical codes (voltammetric signatures) can be readily increased by increasing the number of distinguishable metals or loadings. The new encoded redox beads thus represent a useful addition to the arsenal of particle-based identification tags. The realization of on-site electrochemical authenticity testing should benefit from the availability of portable (hand-held), battery-powered, easy-touse stripping analyzers. Ongoing efforts are aimed at reading the redox label directly on the package and hence making the electrical route competitive with analogous optical avenues. The encoded redox particles also offer great promise for electrical detection of DNA hybridization. ACKNOWLEDGMENT Financial support from the National Science Foundation (Grant CHE 0209707) is gratefully acknowledged. G.R. acknowledges support from NMSU and CONIFET. Received for review April 20, 2003. Accepted June 11, 2003. AC034411K
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