NANO LETTERS
Composite Organic−Inorganic Nanoparticles (COINs) with Chemically Encoded Optical Signatures
2005 Vol. 5, No. 1 49-54
Xing Su,* Jingwu Zhang, Lei Sun, Tae-Woong Koo, Selena Chan, Narayan Sundararajan, Mineo Yamakawa, and Andrew A. Berlin* Biotechnology Research Group, Intel Research and CTM, Intel Corporation, SC2-24, 2200 Mission College BouleVard, Santa Clara, California 95054 Received September 28, 2004; Revised Manuscript Received November 11, 2004
ABSTRACT To obtain a coding system for multiplex detection, we have developed a method to synthesize a new type of nanomaterial called composite organic−inorganic nanoparticles (COINs). The method allows the incorporation of a broad range of organic compounds into COINs to produce surface enhanced Raman scattering (SERS)-like spectra that are richer in variety than fluorescence-based signatures. Preliminary data suggest that COINs can be used as Raman tags for multiplex and ultrasensitive detection of biomolecules.
Multiplex detection is often the method of choice for efficiently deciphering functions or structures of multiple analytes simultaneously in a biological sample. Microarrays1,2 represent one of the great advancements in multiplex detection tools, where two-dimensional coordinates on a solid-phase surface are used to identify a large set of immobilized molecular probes designed to interrogate the mobile phase analytes, such as nucleic acids and proteins. To permit two-phase multiplex reactions (multiplex labeling in both the solid and mobile phases, or in both probes and analytes in the mobile phase), several novel types of labeling materials have been developed. Semiconductor nanocrystals, such as quantum dots3 and submicrometer metallic barcodes,4 which are dependent on physical dimensions, have found various applications in biomedical research.5,6 Raman tags developed by several research groups,7-13 on the other hand, are chemically encoded labeling agents. Because of the richness of Raman signatures and the single-molecule level detection sensitivity14-16 of surface enhanced Raman scattering (SERS),17-20 Raman tags can be used for highly sensitive immunoassays when they are made by coating individual gold particles with extrinsic Raman labels and antibodies.8,13 Recently, several methods for preparing silicacoated Raman tags have been developed,11,12 in which the silica coating makes the Raman tag particles more stable and easier to functionalize. To exploit the potential of SERS technology in multiplex detection, we have developed a new type of Raman tag called composite organic-inorganic nanoparticles (COINs). Unlike Raman tags based on individual gold particles, COINs are * Corresponding authors. E-mail:
[email protected] 10.1021/nl0484088 CCC: $30.25 Published on Web 12/08/2004
© 2005 American Chemical Society
clusters coalesced from metal nanoparticles in the presence of organic Raman labels. Using an organic analyte to aggregate metal particles for SERS detection of the same analyte is known.20 According to theoretical calculations,21,22 a molecule at nanocrystal junctions can produce a Raman scattering intensity several orders of magnitude higher than the same molecule on the surface of single spherical particles. It has also been shown that aggregates of silver or gold nanoparticles are more effective SERS substrates.23-26 Silver nanoparticle clusters capped with conjugated organoisocyanides have been reported to be a molecular sensing mediator via SERS.27 In this work, we demonstrate that a simple and highly scalable procedure can be used to incorporate a variety of organic Raman compounds into COINs to achieve a large number of signatures, and provide evidence to support that Raman labels are embedded at the particle junctions of silver particles to achieve optimal surface enhancement effect, producing spectra similar but not identical to spectra of SERS for the same Raman labels. We provide preliminary data to demonstrate that COINs can be used for multiplex and sensitive bioanalyte detection. As illustrated in Figure 1A, we started with small silver or gold nanoparticles (referred to as seed particles) for COIN synthesis. The silver seeds were prepared by reduction of silver nitrate with sodium borohydride,28 while the gold particles were prepared by reduction of tetrachlorauric acid with sodium citrate.29 The seed particles (equivalent to a metal ion concentration of 0.1 to 0.5 mM) were mixed with known amounts of organic Raman labels (typically in the concentration range from 5 to 30 µM, depending on the
Figure 1. COIN synthesis. (A) Schematic illustration of COIN synthesis and COIN structure, showing particle enlargement, cluster formation, and antibody conjugation. (B) Colorimetric observation during COIN synthesis. A silver seed solution was heated to boil before adding 8-azaadenine (AA) to a final concentration of 20 µM. After 5 min of boiling, additional AgNO3 was added to final concentration of 0.5 mM, then the temperature was lowered and maintained at 95 ( 1 °C. Aliquots (1 mL each) of the solution were retrieved at indicated time intervals (in minutes); the samples were diluted 30-fold with 1 mM Na3-citrate before spectral measurements; the small arrows indicate the characteristic shoulders of the spectra. Inset: photograph of the retrieved sample aliquots (each 50 ul, placed in a Petri dish over a white light box). (C) Time courses of absorptions at indicated wavelengths and Raman signal intensities of sample aliquots. The Y axis values were in arbitrary units after being normalized to respective maxima; the absorbance ratios of 420 nm/395 nm indicate the shift of the main absorption peak from 395 to 420 nm. Raman scattering signals were measured (with a Renishaw Raman spectroscope, see main text) directly from the same diluted samples without using a salt to induce colloid aggregation.
properties of the label molecules), silver nitrate (0.2-1 mM), and sodium citrate (0.2-2 mM). The solution was heated to 95 °C (in a capped bottle) or to boiling (in a reflux apparatus) for 30 to 60 min. During heating, the seed particles grew due to silver deposition and, at the same time, aggregation took place as the adsorption of Raman labels on silver surface decreased the electrostatic repulsion between silver particles. Small aliquots of suspension were withdrawn periodically for particle size and Raman intensity measurements. As soon as the particles reached the desired size (50-100 nm on average), the suspension was cooled to ambient temperature to stop cluster growth. To stabilize COIN samples, bovine serum albumin (BSA, 0.1-0.5% w/v) was added to prevent further aggregation. We used photon correlation spectroscopy (PCS) to monitor the size evolution of COINs and to determine the optimum reaction time. Although SERS-active nanoclusters of metal particles could be formed by aggregating relatively large silver particles (>30 nm) at ambient temperature, the resulting clusters were chemically and physically unstable; for example, such a sample could lose SERS signal when it was exposed to an elevated temperature (>70 °C for 5 min). Therefore, the heating process must have resulted in nanoparticle structures that were different from the structures formed by simple silver particle aggregation. In this report, Raman signals from aqueous samples of SERS and COINs on aluminum surfaces were measured using a Raman spectroscope (Renishaw, UK) equipped with 50
an argon ion laser (514.5 nm, 25 mW), and the data integration time was typically 10 s. The Raman label concentration is another critical parameter for COIN synthesis. The optimum label concentration was determined empirically. If the concentration was too low, there would be no significant cluster formation and the resulting suspension would have very low Raman scattering intensity. In contrast, when the label concentration was too high, rapid aggregation between particles would occur, leading to severe sedimentation. Once the optimum label concentration has been determined, a COIN sample can be reproducibly prepared: typically a standard deviation of less than 15% could be achieved in both Raman signal intensity and average COIN particle size. To gain insight into COIN structures, we monitored color changes of samples during the COIN synthesis with the Raman label of 8-azaadenine (AA). As shown in the inset of Figure 1B, the colors of the silver colloid suspensions changed from yellow to orange, then brown, and finally blue. These color changes indicated the formation of novel particle structures since regular silver suspensions containing dominantly individual colloids look only yellow to gray. We further examined the color changes by absorbance measurements (Figure 1B). The main absorption peak red-shifted from 395 nm to around 420 nm after 50 min of reaction; at the same time, a small shoulder peaked at 500 nm. As the reaction continued, the absorbance at 500 nm decreased and Nano Lett., Vol. 5, No. 1, 2005
Figure 2. Electron micrographs. (A) Transmission electron micrograph (TEM) of silver seed particles. (B) TEM of a crude COIN preparation with single and aggregated silver particles. (C) TEM of a COIN preparation enriched for clusters. (D) Scanning electron micrograph (SEM) of a COIN preparation enriched for clusters.
the absorbance at about 700 nm peaked after about 65 min of reaction. The absorbance at 560 nm by samples retrieved between the two time points was relatively constant. When the samples were analyzed by Raman spectroscopy, we found that the time course and the rates of Raman signal increase (from 40 to 55 min) matched, respectively, with the time course and the rates of the absorption shoulder increase at 560 nm (Figure 1C). In separate experiments, we found that COINs produced stronger signal intensities when they were excited at 514 or 532 nm than at 633 or 785 nm (514 nm ≈ 532 nm > 633 nm > 785 nm). These results suggest that the resonance excitation wavelength for COINs was likely between 500 nm to 600 nm. As seen in Figure 1C, the time courses of the absorption shoulders were polynomial in the ascending phase and their turning points were delayed for the shoulders at higher wavelengths. The above analyses indicate that the COIN synthesis process involves initially silver seed particle enlargement and then particle aggregation or clustering. The morphologies of seed particles and COINs were examined by electron microscopy. As seen in Figure 2A, the seed particles were 2-10 nm (with z-average diameter of 11 ( 1 nm by PCS measurement). In a COIN preparation, enlarged silver particles and coalesced clusters coexisted with seed particles (Figure 2B). To increase the specific activity of COIN (percentage of Raman-active COINs), a large portion of the small particles were removed by pelletting and recovering the COIN particles (>50 nm) using centrifugation (2000× g for 30 min). Very large aggregates (>100 nm) could be removed by low speed centrifugation (
