colloidal silica composite for nucleic acid detection

Journal of Colloid and Interface Science 300 (2006) 117–122 www.elsevier.com/locate/jcis

Facile fabrication of poly(p-phenylene ethynylene)/colloidal silica composite for nucleic acid detection Joong Ho Moon, William McDaniel, Lawrence F. Hancock ∗ Nomadics, Inc., 215 First St., Suite 104, Cambridge, MA 02142, USA Received 13 January 2006; accepted 27 March 2006 Available online 3 April 2006

Abstract Fabrication, characterization, and application of poly(phenylene ethynylene) (PPE)/silica composite particles are described. PPE is a class of conjugated polymers, which has been used for various sensory materials. However, its hydrophobic nature makes its application difficult in the aqueous phase, especially for biological substance detection. In this report, we utilized non-aqueous soluble PPE, 15 nm of colloidal silica particles, and aminosilane to fabricate a biosensory platform. The resulting composite showed high aqueous compatibility, large surface area, high quantum efficiency, and versatile chemical modification including oligonucleotide coupling. By monitoring the fluorescence quenching of PPE, we could detect a quencher-labeled target oligonucleotide specifically. Stern–Volmer (SV) analysis showed different accessibility of fluorophores (PPE) to a quencher labeled target oligonucleotide. The accessibility of fluorophores and SV constant are determined to be 0.54 and 4.2 × 107 M−1 , respectively, from a modified SV plot. This method will broaden the capability of conjugated polymers for the sensitive detection of biological substances. © 2006 Elsevier Inc. All rights reserved. Keywords: Poly(phenylene ethynylene); Nanoparticle; Fluorescence quenching; Stern–Volmer analysis; Nucleic acid detection

1. Introduction Conjugated polymers (CPs) have been used extensively for the sensitive detection of various analytes such as chemicals, metals, and biological substances using fluorescence quenching or fluorescence resonant energy transfer (FRET) [1–5]. The high sensitivity of CPs can be attributed to signal amplification originating from the collective response of molecularly wired fluorophores to analytes [6]. Despite the success of CPs, the hydrophobicity of most CPs complicates their application to the detection of biological substances in aqueous media. By introducing hydrophilic side chains on CPs, researchers demonstrated the detection of oligonucleotides [7] and proteins [8] in water. However, incorporation of charged groups onto a backbone of CPs generally results in a significant loss of luminescence quantum efficiency and increased susceptibility to environmental factors such as pH, temperature, salinity, and surfactant [9]. Therefore, we wished to develop a CP-based * Corresponding author. Fax: +1 617 441 8874.

E-mail address: [email protected] (L.F. Hancock). 0021-9797/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2006.03.063

sensory system showing improved compatibility with aqueous media, while retaining the synthetic versatility and relatively high quantum efficiency of organic-soluble CPs. Here we report a facile fabrication method for a composite of poly(phenylene ethynylene) (PPE) on colloidal silica useful for the detection of oligonucleotides in aqueous media. The composites were constructed by mixing P1, colloidal silica, and an aminosilane in an organic solvent, followed by precipitation in water. The product was silica particles with an ultrathin coating of P1 and amine functional groups on the surface. The composite retained the luminescence quantum yield of the corresponding P1 and has a high surface area. The robustness of the composite allowed for chemical modification in both organic and aqueous solvents. Owing to the hydrophilicity and high specific gravity of silica, covalent attachment of probe oligonucleotides and purification is performed in aqueous media by applying a simple process of centrifugation and decantation. Finally, we demonstrate fluorescence quenching of the probe oligonucleotide grafted composite by specific hybridization of a quencher labeled target oligonucleotide. Details on quencher accessibility to the fluorophores in the complex were also quantified by modified Stern–Volmer analysis.

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2. Materials and methods 2.1. General Chemicals, including solvents, were purchased from Aldrich and used as received. Palladium(0) catalyst was purchased from Strem, and (3-trimethoxysilylpropyl)diethylenetriamine was purchased from Gelest. 20 wt% of colloidal silica dispersed in dimethylacetamide (DMAC) (DMAC-ST) was provided by Nissan Chemical. The mean particle size provided by the manufacturer was 10–15 nm. 3-Maleimidopropionic acid N -hydroxysuccinamide (MPS) was purchased from Molecular Biosciences, Inc. 2XHybridization solution was purchased from Sigma, and aliquots were made by dilution with water (1X). The aliquots were stored at −20 ◦ C until used for hybridization. All oligonucleotides were purchased from Biosource International (CA), and aliquots were made as they were received. The typical concentration of the aliquots was 10−5 M. 5 -SH-CGAGCCTTTACACCGACTCCAACAGCT CG3 (probe), 5 -AGCTGTTGGAGTCGGTGTAAAGGCTCCy5-3 (true target), and 5 -GTC TCA GAC AGT TGT CGCy5-3 (non-target). Purification of the thiol-modified oligonucleotide was carried out by using a NAP5 column according to the manufacturer’s protocol. UV–vis spectra were recorded using a Hewlett–Packard 8453 Diode array spectrophotometer. Fluorescence spectra were obtained using a FluoroMax. The molecular weight of the polymer was determined by gel-permeable chromatography (GPC) using a Plgel 5 µm MiniMIX-C column (Polysciences, Inc.) in 0.1 M LiBr in DMF. The molecular weight is reported relative to PMMA standards purchased from Polysciences, Inc. A transmission electron microscopic (TEM) study was carried out using a JEOL JEM 100 CX scanning transmission electron microscope. TEM samples were prepared by placing a drop of the particle solution on one side of a Cu grid. After a few minutes, excess solution was removed by wicking it away with filter paper. Elemental analysis was carried out by Desert Analytics (Tucson, AZ). A Brunauer–Emmet–Teller (BET) isothermal gas adsorption experiment was performed using an ASAP 2020 accelerated surface area and porosimetry analyzer (Micromeritics, Inc.) under an Ar atmosphere. Particle size was measured by light scattering using a Horiba laser scattering particle size distribution analyzer (LA-910). 2.2. Synthesis P1 was synthesized by a Pd(0)-catalyzed cross-coupling of the pentiptycene diacetylene (2, 1.22 g, 2.55 mmol) and 3 (1.92 g, 2.50 mmol) in a degassed solution of DMF/morpholine (6/4 = v/v) at 80 ◦ C for 16 h. The polymer was recovered by precipitation in methanol, redissolved, and precipitated in acetone to obtain a yellow powder (1.4 g, yield 56%). The molecular weight (Mn ) and polydispersity index (PDI) were estimated by GPC to be 28,000 and 1.6, respectively (PMMA standards in 0.1 M LiBr DMF). 1 H NMR (400 MHz, DMSO-d6): δ 8.47, 8.23, 7.86, 7.72, 7.24, 7.10, 6.84, 5.40, 5.25, 4.16, 4.64, 3.55, 2.37, 2.25, 2.02.

Monomer 3: 18.8 g (31.9 mmol) of 4, 9.3 g (76.4 mmol) of 2-amino-2-(hydroxymethyl)-1,3-propanediol, and an excess (∼20 g) of K2 CO3 were combined with 300 ml of DMSO. The reaction mixture was stirred overnight at room temperature. After filtering off the K2 CO3 , the filtrate (DMSO) was removed by a vacuum distillation. Excess water was added to the resulting viscous oil, resulting in a white powder precipitation. The precipitates were collected by filtration and dried under vacuum. 20.3 g (26.4 mmol, 83%) of white powder was collected. 1 H NMR (400 MHz, DMSO-d6): δ 7.32 (s, –N(CO)H–, 2H), 7.13 (s, ArH, 2H), 4.76 (t, –CH2 OH, 6H), 3.94 (t, Ar– OCH2 –, 4H), 3.52 (d, –CH2 OH, 12H), 2.23 (t, –CH2 C(O)NH–, 4H), 1.68 (m, –CH2 CH2 –, 8H). 13 C NMR (100 MHz, CDCl3 ): δ 173.60, 152.33, 122.39, 86.95, 69.29, 62.28, 60.73, 35.31, 28.15, 21.98. Exact mass ([M + H]): calcd for C24 H38 I2 N2 O10 , 769.0689; found, 769.0688. 2.3. Fabrication of P1/silica composite 0.2 ml of 20 wt% colloidal silica sol in DMAC, 2 ml of DMAC, and 0.1 ml of NH4 OH were combined and stirred rapidly for 30 min. 2.2 ml of P1 dissolved in DMAC (1 mg/ml) was transferred into the silica solution, followed by the addition of 0.1 ml of 3-(trimethoxysilylpropyl)ethylenetriamine. The solution became slightly viscous after 30 min of stirring at room temperature, and then the solution was transferred into 6 ml of water with rapid stirring. Precipitates were collected as a pallet from centrifugation and decantation, and the pallet was resuspended into DMAC. The composite in DMAC was centrifuged again, and yellowish supernatant was discarded. Various organic solvents including DMF, DMSO, and N -methyl 2-pyrrolidinone (NMP) were used to further wash off unreacted PPE from the composite. This washing step was repeated (usually two to three times), until no color was observed from the supernatant. The composite particle was then washed with water three times and stored in water for the oligonucleotide coupling. The composite was lyophilized for quantification, resulting in an off-yellow powder (48 mg). 2.4. Activation of the composite and probe oligonucleotide coupling For the efficient coupling chemistry of MPS with amine, we changed the solvent from water to DMF by centrifuging/decanting (three times). 1 ml of the composite in DMF and ∼10 mg of MPS were combined in a 1.5 ml centrifugal tube, and agitated in a Fisher Minishaker set at 1400 rpm for 1.5 h. Excess MPS was removed by rinsing with 1 ml of fresh DMF five times, and the supernatant was discarded. The maleimide-activated composite particle in ∼0.03 ml of residual DMF was transferred into a 0.9 ml of desalted oligonucleotide solution. The oligonucleotide was desalted using a NAP5 column as directed by the manufacturer’s manual. The solution was placed in the shaker overnight. The probe-attached composite was thoroughly cleaned by centrifugation with 1 ml of 0.1% SDS (three times) and decantation with fresh water (three

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times). The final particle was stored in ∼0.1 ml of water, and used for the hybridization experiment. 2.5. Hybridization 20 µl of probe-attached particle was suspended in 1.5 ml of 1XHybridization solution, and shaken for 1 h. The solution was then transferred into a 1 ml disposable polystyrene cuvette (Aldrich) for a fluorescence measurement. After collection of the reference fluorescence spectrum excited at 415 nm, a quencher-labeled oligonucleotide in 1XSSPE (saline sodium phosphate EDTA) (or water) was added by pipette. Typically, 10–20 µl of 10−5 M quencher-labeled oligonucleotide was added. The fluorescence spectrum was collected and compared immediately after the addition of the target oligonucleotide. For the non-target control experiment, the same procedures were carried out in parallel using a quencher-labeled non-target. 3. Results and discussion 3.1. Fabrication and characterization of PPE/silica composite P1 was polymerized by standard palladium catalyzed crosscoupling of the pentiptycene diacetylene and bis(dialkoxy)diiodobenzene monomer (Scheme 1). The resulting polymer was soluble in DMF, DMSO, DMAC and NMP, and was not soluble in methanol and water. UV absorption and fluorescence emission spectra showed typical absorption (with maximum at 450 nm) and sharp emission (at 460 nm) of PPEs. We incorporated the rigid three-dimensional pentiptycene units to the polymer backbone, because it minimizes chain–chain interaction (π –π stacking) in the solid state (film on silica surface) that would otherwise lead to fluorescence self-quenching. To exploit the outstanding photophysical properties of the pentiptycenecontaining polymers for the detection of analytes in aqueous phase, we polymerized the pentiptycene with a diiodo monomer that contains tris(hydroxymethyl)aminomethane (TRIS) units

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(Scheme 1, 3). The TRIS units help the solubility of P1 in polar solvents and provide functional groups to interact with silanols from the colloidal silica surface. A PPE/silica composite was fabricated by mixing P1, colloidal silica, and (3-trimethoxysilylpropyl)diethylenetriamine in DMAC, followed by the precipitation of the mixture in basic water (details in experimental procedures). The adsorption mechanism of P1 onto the colloidal silica particles is not clear, but is likely hydrogen bonding of P1 to the surface of the silica particles in the reaction media. Subsequently, the aminosilane induced a strong ionic interaction among the colloidal silica particles resulting in the formation of the polymer (P1)/silica composites. The composite was further stabilized by the hydrolysis and condensation of the aminoalkoxysilanes during the precipitation in alkaline solution [10]. The aminosilane was introduced at the end of fabrication procedure to fully utilize amine groups for the conjugation of probe oligonucleotides. The precipitates were washed thoroughly with various solvents such as DMAC, DMF, DMSO, acetone, and water to remove any unreacted materials from the reaction mixture. Spectroscopic studies revealed that the resulting composite particles exhibited UV absorption and luminescence similar to P1 in organic solvent with a peak centered at 450 nm (Fig. 1) and a sharp emission band at 460 nm (see Fig. 3). The composite retained its sharp spectral features in both NMP and water similar to the P1 in NMP (Fig. 1), indicating that the P1 maintained the original polymer’s conformation such as conjugation length during adsorption onto the silica surface. The absorption spectrum of P1 in a poor solvent (water) was smoothed, and the absorption maximum moved to short wavelength (hypsochromic shift). This spectral change was initiated by collapsing polymer chains to form spherical particles [11], resulting in a shortened conjugation length. Since the composite showed the similar spectral shape in any solvents including water, it is likely that the P1 experiences minimal structural reorganization during adsorption onto the silica. In addition, little spectral change was observed upon the addition of a surfactant such

Scheme 1. Synthesis of monomers and P1.

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Fig. 2. Transmission electron microscopic image of a P1/silica composite. Inset is an enlargement of a selected area.

Fig. 1. UV–vis spectra of (a) P1 and (b) P1/composite in NMP (1), water (2), and SDS/water (3). While absorption feature of P1 changed upon solvents, the composite retained identical absorption characteristics to the corresponding P1.

as sodium dodecyl sulfate (SDS). Had the PPE been weakly bound onto the silica surface, SDS would have caused reorganization such as planarization in PPEs and thus a spectral change [12]. Indeed, the addition of SDS to an aqueous solution of P1 changes the UV spectrum to one that is similar to P1 in NMP, with its characteristic sharp peak at 450 nm (Fig. 1a). The spectral noise and shoulder around 500 nm in Fig. 1b indicate increasing particle size from the aggregation of the composite particles, resulting in scattering UV–vis light and the bathochromic shift due to extra interaction between composites. We found no loss of quantum efficiency from the adsorption of P1 onto a silica particle: the fluorescence quantum yield (0.42) of P1 in NMP was preserved even after composite formation in NMP (0.48) [13]. A transmission electron microscopic (TEM) image (Fig. 2) shows the aggregated composites in aqueous media. The inset of Fig. 2 indicates that individual silica particles were aggregated with clear morphology of silica particles. The average size of the aggregates in water was measured by light scattering, showing median diameter of 22–26 µm. The size of aggregates in water was reduced dramatically to about 100 nm by applying shear forces at high pressure [14]. This indicates that the composite was physically aggregated due to the hydrophobic nature of P1 in water. We are currently investigating to find a condition for fabricating small and uniformly sized particles. Elemental analysis implied the ultrathin coating layer. The amount of P1 in the composite was very low (3.5 wt%), compared to the silica (SiO2 , 85 wt%) [15]. A BET isothermal gas adsorption experiment indicated that the dried composite has a surface area of 149 m2 /g, which is quite high [16]. The ultrathin coating of sensory materials combined with a high surface area should play an important role in improving sensing efficiency.

Wang et al. reported that an enhancement of two to three orders of magnitude in the SV constant was observed when sensory materials were fabricated with a high surface area-to-volume ratio compared to a film format [17]. The high surface area contributes to high sensitivity to quenching analytes due to both a higher density of binding sites, and a lower background fluorescence signal. 3.2. Specific hybridization of PPE/silica composite for a labeled target oligonucleotide For detection of oligonucleotides, we modified the amine rich composite with a MPS in DMF (Scheme 2). The maleimide-functionalized particles were then reacted with a thiolmodified oligonucleotide probe in water overnight (see details in Section 2). Because of the high specific gravity of the colloidal silica, a mild centrifugation (<10 s at 5–6 K g-forces) was sufficient to isolate the oligonucleotide-labeled particles from the reaction solution. The particles were resuspended in a hybridization buffer (Sigma) containing either a Cy5-labeled complementary oligonucleotide (“true target”) or a Cy5-labeled non-complementary oligonucleotide (“non-target”) at room temperature. Cy5 is commonly used as a fluorophore in gene expression analysis; we used it as a quencher in this work [11]. Fig. 3 shows the change in fluorescence of the composite upon hybridization with a complementary target oligonucleotide (a) and a non-complementary target oligonucleotide (b). Strong fluorescence quenching was observed with the complementary oligonucleotide, while the non-complementary oligonucleotide induced little change in the fluorescence intensity even at higher concentration (trace 4 in Fig. 3 was obtained by direct excitation of the Cy-5 dye at 635 nm) [18]. In this experiment, the original fluorescence intensity was reduced by 61% at 5 × 10−7 M of true target concentration. Quenching is observed immediately upon addition of the target sequence (trace 2 in Fig. 3). Hybridization was complete within an hour at high target concentration (>10−7 M) (trace 3 in Fig. 3); a longer time was required at lower target concentration (e.g., 10−9 M).

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Scheme 2. Schematic drawing of composite modification for probe oligonucleotide coupling and its specific hybridization with a quencher-labeled target oligonucleotide.

Fig. 3. Fluorescence spectra of unlabeled probe oligonucleotide attached composite before (solid) and after hybridization (broken). Fluorescence intensity was monitored after incubation with (a) Cy5-labeled true target and (b) Cy5-labeled non-target, respectively. (1) Initial, (2) right after hybridization, (3) 1-h hybridization, and (4) emission of Cy5 by direct excitation at 635 nm.

3.3. Stern–Volmer analysis Analysis of the SV plot is useful to determine the fluorophore accessibility to the quencher as well as to estimate the binding constant [19]. An SV plot from a specific hybridization is shown in Fig. 4. Data points for the SV plot were taken after 1-h hybridization. Downward curvature in Fig. 4 implies the existence of different surface sites in the population of the composite where access to the fluorophore (PPE) by the quencher labeled oligonucleotides varies. Considering the aggregated structure, some P1s are located inside of the composite, which are not accessible for the fluorescence quenching. Unfavorable geometry of probes on the surface can be another reason. Duplex formation will be more effective when the probe oligonucleotide has excluded volume for hybridization with a target oligonucleotide. Densely packed probes on the composite surface could result in poor hybridization efficiency—i.e., sites inaccessible to the quencher. Indeed, quenching efficiency was almost doubled when the probe density was decreased by half [20].

Fig. 4. Stern–Volmer (SV) plots from specific hybridization. Downward curvature indicates different accessibility of fluorophores to a quencher. The accessibility (fa ) was calculated from the modified SV equation, showing accessibility of 0.54. Curve fitting was performed by the modified SV equation [19].

In the inset of Fig. 4, the data is replotted using the modified Stern–Volmer equation   F0 /(F0 − F ) = 1/fa Ka [Q] + 1/fa , (1) where F0 and F are the total fluorescence in the absence and presence of the quencher, respectively, fa is the fraction of the initial fluorescence that is accessible to the quencher, Ka is the SV constant of the accessible fraction, and [Q] is the quencher concentration. From the intercept (fa−1 ) and slope ((fa Ka )−1 ), the fraction of the accessible fluorophores and SV constant are determined to be 0.54 and 4.2 × 107 M−1 , respectively. In summary, we have demonstrated a novel fabrication method for the production of PPE/silica composite, and applied this functionalized material to the detection of specific oligonucleotides. Since we used non-water soluble PPE to make this biosensory material, this method will enhance the capability of CPs to detect of biological compounds including nucleic acids, proteins, and pathogens. Reducing the size of the composite to nanometer scale should increase the practical utility of this material in a gene expression system and quantitative polymerase chain reaction.

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Acknowledgment The authors thank the National Science Foundation (SBIR Grant DMI-0239285) for support of this work.

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(b) M. Levitus, K. Schmieder, H. Ricks, K.D. Shimizu, U.H.F. Bunz, M.A.G. Garibay, J. Am. Chem. Soc. 123 (2001) 4259. QY was measured using 9,10-diphenylanthracene in cyclohexane as a reference. QY of the composite in water could not be determined due to the interference of light scattering from aggregated particles in water. The composite was passed through a commercially available chamber (Microfluidizer M-110S, Microfluidics, Newton, MA) at 23,000 psi. The system utilizes high-pressure streams that collide at ultrahigh velocities in precisely defined microchannels. Total weight fraction of P1 was estimated based on elemental analysis results (% C: 7.12, H: 1.70, N: 2.29, and Si: 41.22). As a control, we fabricated a aminosilane/silica material with no P1. Elemental analysis indicated relatively low carbon (5.35%) and nitrogen (1.70%) content compared to the original P1/silica composite. Our calculated weight fraction of aminosilane based on the amount of nitrogen in the elemental analysis was 8.9–10.7 wt%. The calculated amount of aminosilane in the control material was well in accordance with the aminosilane fraction of the P1/silica composite (8.5–11.5 wt%). Calculated surface area of the 1 g of 14 nm silica particles is 199 m2 /g. X. Wang, Y.-G. Kim, C. Drew, B.-C. Ku, J. Kumar, L.A. Samuelson, Nano Lett. 4 (2004) 331. We also modified the composite particle with a 26-mers probe (5 -SHGCGACTAACGTCAATTGCTTTGTCGC-3 ), and performed hybridization with same length of a true target (20-mers, 5 -GACAAAGCAATT GACGTTAG-BHQ1-3 ) and a false target (20-mers, 5 -GGTCTTTCT GCATTCCTGGA-BHQ1-3 ), respectively. Fluorescence quenching was observed from the true target (53% at 1 × 10−7 M), while the false target at the same concentration produced only 4% quenching. We compared quenching ability of Cy5 and BHQ1 and found no significant difference between them. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic/Plenum, New York, 1999. We adjusted the probe density by transferring maleimide functionalized complex into a mixed solution of thiol-modified oligonucleotide: random 12-mers and specific 29-mers (1:1 molar concentration). Fast hybridization at the composites with adjusted probe density results in increased fluorescence quenching within the hybridization time. Details on the probe density effect on the quenching efficiency will be published soon.