19604
J. Phys. Chem. B 2005, 109, 19604-19612
Homogeneous, Competitive Fluorescence Quenching Immunoassay Based on Gold Nanoparticle/Polyelectrolyte Coated Latex Particles Noritaka Kato† and Frank Caruso* Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The UniVersity of Melbourne, Victoria 3010, Australia ReceiVed: May 25, 2005; In Final Form: August 4, 2005
This study reports a homogeneous and competitive fluorescence quenching immunoassay based on gold nanoparticle/polyelectrolyte (AuNP/PE) coated latex particles prepared by the layer-by-layer (LbL) technique. First, the resonant energy transfer from a layer of fluorescent PEs to AuNP in LbL assembled films on planar substrates was investigated. The quenching efficiency (QE) for the planar films depended on the cube of the distance between the two layers. A QE of 50% was achieved at a distance of ca. 15 nm, indicating that the AuNP/PE system is suitable for detecting binding/release events for antibodies. A homogeneous, competitive binding immunoassay for biotin was designed based on AuNP/PE-coated polystyrene particles of 488 nm diameter as quenching agents for a fluorescein isothiocyanate labeled anti-biotin immunoglobulin (FITCanti-biotin IgG). Biotin molecules were localized on the AuNP/PE-coated latexes by depositing a layer of biotinylated poly(allylamine hydrochloride) (B-PAH), and FITC-anti-biotin IgGs were subsequently bound to the particles through interaction with the biotin on B-PAH. Transmission electron microscopy and quartz crystal microgravimetry confirmed the multilayer formation on latex particles and planar gold surfaces, respectively. The biotin-functionalized AuNP/PE-coated latexes terminated by FITC-anti-biotin IgG exhibited a dynamic sensing range of 1-50 nmol. These results indicate that AuNP/PE-coated latexes can be readily used as dynamic range tunable sensors.
Introduction There have been a number of developments in fluorescence immunoassays (FIAs), and numerous fluorophores and methods have been proposed.1-3 A promising approach is based on fluorescence quenching immunoassays (FQIAs), where extensive studies on fluorescence quenching by metals have been conducted.4-12 When a fluorophore is close to a metal surface, energy transfer (ET) from the fluorophore to the metal occurs, resulting in fluorescence quenching. This allows monitoring of receptor/ligand binding and release events through changes in the fluorescence intensity or lifetime of the fluorophore. Metal films deposited on planar substrates and dispersed metal (gold and silver) nanoparticles have been applied as quenchers in FQIAs.4-12 Surface plasmon (SP) modes have been utilized to effect resonant ET or excitation enhancement, resulting in a higher degree of quenching or emission, reflecting binding and unbinding states of analytes to sensors. Despite such studies, metal nanoparticles supported on colloidal submicron-sized particles have not been applied in FQIAs. Compared with metalcoated planar films or nanoparticles, the application of colloidally stable nanoparticle-coated submicron-sized colloids in FQIAs offers the combined advantage of both methods: the ability to tailor the surface functionality to the same level as for planar substrates, high surface area for analyte binding/ release, and ease of handling, as the particles can be readily collected through filtration and/or centrifugation, with the potential for reuse. Further, the surface area of the colloidal * Corresponding author. E-mail:
[email protected]. † Present address: Department of Physics, Waseda University, Tokyo 169-8555, Japan.
dispersion can be easily altered by changing the particle concentration, allowing tuning of the dynamic sensing range. Recently, we showed that gold nanoparticle (AuNP) coated particles with tailored optical properties and nanoparticle loadings could be formed by the layer-by-layer (LbL) technique.13-18 The particles were prepared by the sequential deposition of oppositely charged polyelectrolytes (PEs) onto colloidal particles, followed by adsorption of ligand-stabilized gold nanoparticles into the PE multilayer films. We applied such particles in photonic crystal studies to modulate the optical properties of colloidal crystals,13-15 and as optically addressable capsules, which can release (bio)macromolecules upon irradiation with near-infrared light.16,17 In this study, we design and tailor AuNP/PE-coated latex particles (AuNP/PE latexes) for use as quenching agents in FQIAs. The AuNP/PE latexes were prepared by depositing AuNP on submicron-sized latex spheres precoated with PE multilayers.13,18 The SP band of the AuNP on the latexes is utilized for the resonant quenching of fluorophores coupled to antibodies, which are attached to the surface of the particles (see Figure 1). When bound on the surface of the AuNP/PE latexes, the fluorescence from the antibodies is quenched due to ET (Figure 1a). After injection of the analytes in the suspension (Figure 1b), the antibodies are competitively released from the AuNP/PE latexes into solution and became fluorescent (Figure 1c), resulting in a homogeneous and competitive binding immunoassay. We demonstrate a FQIA based on the AuNP/PE latexes by using biotin molecules as analytes, and fluorescein isothiocyanate (FITC) conjugated anti-biotin immunoglobulin (FITC-antibiotin IgG) as fluorophores (Figure 1).10 Before preparation of the functionalized latexes, we investigated the quenching efficiency between AuNP and FITC layers to examine the
10.1021/jp052748f CCC: $30.25 © 2005 American Chemical Society Published on Web 10/01/2005
FQIA Based on AuNP/PE-Coated Latex Particles
J. Phys. Chem. B, Vol. 109, No. 42, 2005 19605
Figure 2. Molecular structures of materials used for biotinylation of PAH. (a) PA, (b) biotin-ANHS, and (c) B-PAH.
Figure 1. Schematic illustration of competitive FQIA using a suspension of AuNP/PE-coated latexes. (a) Fluorophore-labeled antibodies are quenched before injection of analyte (biotin). (b) Injection of analyte. (c) Fluorophore-labeled antibodies fluoresce after being competitively released by biotin.
properties of resonant ET. This was studied in planar films by changing the amount of AuNP in the layer and the distance between the AuNP and FITC layers. For biotin localization on the AuNP/PE latexes, biotinylated poly(allylamine hydrochloride) (B-PAH) was prepared, and the binding specificity of the B-PAH was confirmed by constructing avidin/B-PAH multilayers on planar supports. The fluorescence intensity of the latex dispersions as a function of analyte concentration was monitored to evaluate the dynamic sensing range for biotin. Experimental Section Materials. Poly(sodium 4-styrenesulfonate) (PSS) (Mw ) 70 000), poly(ethylene imine) (PEI) (Mw ) 25 000), PAH (Mw ) 15 000 and 70 000), FITC, dimethyl sulfoxide (DMSO), 4-(dimethylamino)pyridine (DMAP), gold tetrachloride (HAuCl4), sodium borohydride (NaBH4), toluene, 20 wt % aqueous solution of poly(allylamine) (PA) (Mw ) 17 000), biotinami-
dohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (biotin-ANHS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), bovine serum albumin (BSA), 2-(4′-hydroxyazobenzene)benzoic acid (HABA), lyophilized powder of avidin, and FITC-conjugated avidin (FITC-avidin) from egg white were purchased from Sigma-Aldrich. Tetraoctylammonium bromide (TOAB), hydrochloric acid (HCl), sodium chloride (NaCl), and sodium hydroxide (NaOH) were from Fluka. Biotin and the FITC-conjugated goat polyclonal anti-biotin immunoglobulin (FITC-anti-biotin IgG) (1 mg mL-1 aqueous solution) used for the competitive FQIA were purchased from Sigma-Aldrich. An aqueous HEPES solution (0.01 M, 0.1 M NaCl, pH 7.3 ( 0.1) was used as buffer. For the anti-biotin antibody, the HEPES buffer containing 1 wt % BSA was used to reduce the nonspecific binding. Before use, the buffers were filtered (Minisart, 200 nm pore size, Sartorius). Polystyrene (PS) latexes with a diameter of 488 nm and sulfonated surfaces were purchased from Microparticles GmbH (Berlin, Germany). Synthesis of AuNP Dispersion. An aqueous suspension of AuNP with an average diameter of ca. 6 nm was prepared, as reported previously.19 A 30 mL volume of aqueous HAuCl4 (10 mg mL-1) and 80 mL of TOAB in toluene (25 mM) were mixed to transfer the gold salt to the toluene phase. The toluene phase was washed with water until the pH of the separated water became neutral; then 10 mL of aqueous NaBH4 (30 mg mL-1) was slowly added to the separated toluene with stirring. This toluene/aqueous solution was then incubated overnight without stirring, and AuNP were formed in the toluene phase. The toluene phase was again washed with water until the pH of the separated water became neutral. The AuNP were then transferred from toluene to an aqueous DMAP solution by adding 0.1 M DMAP, resulting in a DMAP-stabilized AuNP dispersion, with a SP peak at approximately 518 nm. Polymer Labeling. FITC-labeled PAH (FITC-PAH) was prepared by a method based on a protein labeling.20,21 A 0.42 mL volume of FITC in DMSO (10 mg mL-1) was added to 25 mL of aqueous PAH (Mw ) 15 000) (4 mg mL-1, pH adjusted to 9.0 by 1 M aqueous NaOH) and gently stirred overnight. The resulting FITC-PAH was then purified by a Slide-A-Lyzer dialysis cassette (3500 MWCO, 3-12 mL capacity, PIERCE). The dialysis was carried out in darkness against pure water for 48 h, renewing the water four times. The degree of the labeling was ca. 1% PAH monomer.21 B-PAH was obtained by following a procedure based on reported methods.22,23 The molecular structures of the materials used are given in Figure 2. A 50 mg sample of 20 wt % aqueous PA and 4 mg of biotin-ANHS were added to 8.5 mL and 1.5 mL of 0.2 M bicarbonate buffer (pH 8.3), respectively. The
19606 J. Phys. Chem. B, Vol. 109, No. 42, 2005 solutions were mixed together and stirred overnight to achieve randomly labeled PA with biotin. The stirred solution was then injected into the dialysis cassette (3500 MWCO, 3-12 mL capacity, PIERCE) and dialyzed against water for 96 h. Water for the dialysis was renewed on a 24 h cycle. Pure water was used for the first cycle. For the remaining cycles, water of pH 4, prepared by adding aqueous HCl, was used to convert the amine groups to ammonium groups for improved solubility. After the solution was removed from the dialysis cassette, it was filtered (Minisart, 200 nm pore size, Sartorius) to remove the aggregated PEs, resulting in ca. 50% yield. To evaluate the degree of the labeling, the solvent of the obtained B-PAH solution was exchanged into heavy water and analyzed by nuclear magnetic resonance (NMR). Using the integrated values of two peaks at 4.4 and 4.2 ppm in the proton NMR spectrum, which originate from the two protons of biotin (see the marked protons in Figure 2c),24,25 the labeling was calculated to be ca. 4% of the monomer units in the polymer. LbL Coating of Planar Supports and Microspheres. The procedure and conditions used for preparing LbL films are as follows. The polyanion adsorption solution was PSS (1 mg mL-1 with 0.5 M NaCl), and the polycation solutions were PEI or PAH (Mw ) 70 000) (1 mg mL-1 with 0.5 M NaCl). Quartz or glass slides were used as negatively charged substrates after RCA cleaning.26 The layers were deposited by (i) dipping the glass substrates in the adsorption solution and incubating for a given time, (ii) removing the coated substrate from the adsorption solution and rinsing with a washing solution (e.g., water), and (iii) drying the coated substrate using compressed air. The adsorption time depended on the sample (typically 10 min). In the case where drying after layer deposition was not desired, step iii was omitted. Repetition of these steps, alternating the polycation and polyanion adsorption solutions, resulted in the buildup of PE multilayer thin films. The same adsorption solutions used for the planar substrates were employed for the microspheres (PS latexes). A 25 µL volume of a 1 wt % aqueous suspension of the PS latexes was transferred to a 2 mL centrifuge tube (Eppendorf) and washed with 1.5 mL of pure water three times by centrifugation/water wash cycles. The polycation was then deposited as the first layer because of the negatively charged surface of the PS latexes. For each layer, the following steps were performed: (i) centrifuge (10000g for 12 min) the suspension and remove the supernatant; (ii) disperse the sediment using brief sonication (
