Polycyanurate Nanorod Arrays for Optical-Waveguide-Based

pubs.acs.org/NanoLett

Polycyanurate Nanorod Arrays for Optical-Waveguide-Based Biosensing Antonis Gitsas,† Basit Yameen,‡,| Thomas Dominic Lazzara,‡,⊥ Martin Steinhart,§ Hatice Duran,*,‡ and Wolfgang Knoll*,† †

Austrian Institute of Technology GmbHsAIT, Nano Systems, Donau-City-Strasse 1, 1220 Vienna, Austria, Max-Planck Institut fu¨r Polymerforschung, 55128 Mainz, Germany, and § Universta¨t Osnabru¨ck, Institut fu¨r Chemie, 49069 Osnabru¨ck, Germany ‡

ABSTRACT We demonstrate high-sensitivity biosensing by optical waveguide spectroscopy (OWS) at visible wavelengths using aligned polycyanurate thermoset nanorods (PCNs) arranged in extended arrays as waveguides. The PCNs formed by thermal polymerization of a cyanate ester monomer in self-ordered nanoporous alumina templates were 60 nm in diameter and 650 nm in length. Subtle refractive index changes of the medium surrounding the nanorods could be detected by monitoring the angular shifts of waveguiding modes. The sensing figure of merit thus achieved amounted to 196 reciprocal refractive index units and is, therefore, higher than that of other sensors based on angular modulation, while the configuration used here is eligible for further surface functionalization. Kinetics of the binding of taurine to the surface cyanate groups of the PCNs was monitored by OWS. Thus, modified PCNs bearing sulfonic acid groups at their surfaces were obtained. PCN arrays may represent a versatile platform for the design of biosensors. KEYWORDS Waveguide, nanorod array, biosensor, anodic alumina, taurine

T

he fabrication of one-dimensional nanostructures by replicating shape-defining nanoporous templates has become a well-established synthetic methodology.1,2 Self-ordered anodic aluminum oxide (AAO)3 containing arrays of aligned cylindrical nanopores characterized by narrow pore diameter distributions is a template system widely used to this end. Functionalized AAO membranes and other inorganic nanoporous scaffolds were employed as optical waveguides for visible light4 and for various sensing applications.5-8 Moreover, biosensing with plasmonic gold nanorod metamaterials obtained by electrodeposition into AAO was demonstrated.9 However, the templatebased fabrication of released nanorod arrays stable under real-life conditions is still challenging.10 Furthermore, the integration of optically transparent nanorod arrays into device architectures enabling optical waveguide spectroscopy (OWS) studies has, up to now, not been accomplished. OWS, which is based on evanescent wave optics and the monitoring of waveguide modes, has been employed to characterize optical properties of thin films.11,12 Here we show that polycyanurate thermoset nanorod (PCN) arrays are efficient and sensitive waveguide sensors that can be operated with visible light. The PCNs with a length of 650 nm have a diameter of 60 nm, which is smaller than one-

tenth of the wavelength of the guided light. Thus, waveguiding along the PCNs results in pronounced coupling of the guided waves to the PCN surfaces as well as to the volume in between of the PCNs. Hence, minute changes in the refractive index of the medium surrounding the PCNs or changes in the effective refractive index of the detection volume, neff, caused by binding of analytes to the surfaces of the PCNs are amplified. Solvents with slightly different refractive indices surrounding the PCNs could easily be distinguished by OWS. Moreover, by OWS covalent attachment of taurine to the surfaces of the PCNs could be monitored in situ. A schematic diagram illustrating the fabrication of PCNs attached to Au substrate and the OWS setup is shown in Figure 1. At room temperature, self-curing cyanate ester monomers (CEMs)13 are low-viscous liquids, which can easily be infiltrated into AAO. The PCNs obtained by cross-linking CEMs (Figure 1a) have high chemical and mechanical stability (Tg ) 600 K; Young’s modulus ∼3 GPa) as well as a high permittivity (ε′ ) 3.62 at f ) 1 MHz; 25 °C)13 related to the polarizable aryl ether linkage. Most importantly, PCNs have cyanate groups on their surfaces that allow further chemical functionalization. The PCNs were prepared as follows: A 2 nm thick Cr film and subsequently a 50 nm thick Au layer were deposited on a high refractive index glass slide (LaSFN9, Hellma Optik, n ) 1.845). The gold surface was aminofunctionalized by dipping the specimen into a solution of 5 mM 2-aminoethanethiol in absolute ethanol (Sigma-Aldrich) for 45 min. The CEMs13 were infiltrated by spin coating (3000 rpm, 2 min) into AAO (pore diameter 60 nm, pore depth 650 nm, lattice constant 105 nm) connected to an underlying Al substrate and kept under vacuum (5-10

* Corresponding authors, [email protected] and wolfgang.knoll@ ait.ac.at. |

Current address: Faculty of Materials Science and Engineering, GIK Institute of Engineering Sciences and Technology, Topi (23640), NWFP, Pakistan.

⊥

Current address: Institute for Organic and Biomolecular Chemistry, GeorgAugust Universita¨t Go¨ttingen, Tamann Str. 2, 37077 Go¨ttingen, Germany. Received for review: 3/15/2010 Published on Web: 05/18/2010 © 2010 American Chemical Society

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FIGURE 1. Preparation of PCNs and PCN-based OWS setup. (a) Generalized scheme of thermal curing by cyclotrimerization of the cyanate groups in cyanate ester monomers yielding thermosetting polycyanurate. (b) Infiltration of CEMs into AAO. (c) Attachment of CEM-infiltrated AAO to a LaSFN9 glass slide covered with a cysteamine-functionalized Au layer. The open ends of the pores of the AAO pointed toward the LaSFN9 glass slide. (d) Bird’s eye view scanning electron microscopy image of an array of released PCNs oriented normal to the LaSFN9 glass slide (scale bar is 1 µm). (e) PCN array integrated in an OWS setup. The LaSFN9 glass slide with the PCN array is attached on a LaSFN9 prism. The laser light entering the prism at an incident angle θ had either transversal magnetic (TM) or transversal electric (TE) polarization.

tion on a customized setup8,12 (Figure 1e). Laser light (HeNe, λ ) 632.8 nm) was passed through a chopper and two polarizers before being incident on one face of a LaSFN9 prism (Schott Glass). The first polarizer was used to adjust the intensity of the incident light and the second one to adjust the polarization of the electric field either parallel (transverse magnetic, TM) or perpendicular (transverse electric, TE) to the propagation plane (Figure 1e). TM modes correspond to electron excitations along and TE modes to electron excitations normal to the long axis of the PCNs, respectively. The sample was attached on the prism with index matching fluid (Cargille Laboratories Inc., n ) 1.7000 ( 0.0002). A poly(tetrafluoroethylene) flow cell with a volume of 109 µL was mounted on top of the PCN array. The precise angle of incidence of the light was controlled with a computer-operated goniometer, while the intensity of the reflected light was measured with a photodiode. The PCN array acts as waveguiding core, whereas the underlying Au layer (ε* ) (-12) + 1.2i) and the medium surrounding the PCNs (air with εair ) 1.000 or solvents) act as cladding. The PCN array and the medium surrounding the PCNs were considered as an effective medium with effective refractive

mbar) at 120 °C for 12 h (Figure 1b). After removal of excess CEMs from the surface of the AAO, the CEM-filled AAO was pressed against the amino-functionalized gold-coated surface of the glass slide and thermally cured (Figure 1c) under nitrogen atmosphere by heating to 120 °C for 1 h, to 180 °C for 8 h, to 260 °C for 3 h and to 290 °C for 1 h. The PCNs thus obtained were connected to a ∼20 ( 5 nm thick polycyanurate film covalently bonded to the amino-functionalized Au surface of the glass substrate. The Al substrate was then dissolved with aqueous CuCl·2H2O/HCl solution at 0 °C, and the AAO with 10% aqueous H3PO4 solution at room temperature for 12 h. As a result, an array of freestanding PCNs oriented normal to the Au-coated surface of the glass slide extending ≈3 cm2 was obtained (Figure 1d). For OWS, a detection area corresponding to the cross section of the incident laser beam of ≈1 mm2 is sufficient. Several OWS measurements at different positions on the PCN array yielded identical results, thus evidencing its homogeneity. It is, therefore, conceivable that PCN arrays could be used as components in OWS sensor arrays. Optical waveguide spectroscopy was measured in the Kretschmann attenuated total internal reflection configura© 2010 American Chemical Society

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index neff and effective permittivity εeff weighted by the volume fractions of the components, assuming the permittivity of the bulk polycyanurates to be 3.6213 and taking into account the free-standing nanorod geometry of the array for the calculations.4,8,14 The anisotropic dielectric response of the PCN arrays may be described by the infinite, prolate ellipsoid approximation of the Maxwell-Garnett theory14-18

⊥ εPCN

1 (1 + fPCN)(εPC - εS) 2 ) εS 1 εS + (1 - fPCN)(εPC - εS) 2

(1a)

|| εPCN ) εS + fPCN(εPC - εS)

(1b)

εS +

⊥ || Here, εPCN and εPCN are the permittivities of the sensor normal and parallel to the PCNs long axis, εPC is the permittivity of the polycyanurates, fPCN is the volume fraction of the PCNs, and εS is the permittivity and of the medium surrounding the PCNs. Since the diameter of the PCNs, DPCN, amounted to 60 nm and was, therefore, less than 1/10 of the wavelength of the incident light (λ ) 632.8 nm), the “quasistatic” limit of the effective medium theory approximation (EMT) in which dielectric response induced by the incident field is in phase over the entire nanodomain could be assumed.4,15 In the course of the OWS measurements, the intensity of the reflected beam, R, was measured as a function of the incidence angle of the incoming beam, θ, in the range 20-70° with an angular resolution of 0.01°. Coupling of the incident beam to the PCN array accompanied by guiding of modes through the PCNs is apparent as minima of the reflectivity. Changes in εeff result in an angular shift ∆θ of the reflectivity minima belonging to specific modes propagating through the PCNs. To evaluate the sensitivity with which refractive index changes in the immediate environment of the PCNs can be detected by OWS, we conducted solvent exchange experiments using deionized water (n ) 1.333, ε ) 1.777) and isopropanol (n ) 1.378, ε ) 1.899) as model liquids with similar n and ε values. Two guided optical modes θm1 and θm2 were observed for both systems PCNs/water and PCNs/ isopropanol (Figure 2). Since the refractive index of isopropanol is higher than that of water, the corresponding effective permittivity increases from εeff,water ) 2.360 for the system PCNs/water to εeff,isoprop. ) 2.405 for the system PCNs/ isopropanol. As a result, angular shifts of ∆θm1,TM ) 1.57° and ∆θm2,TM ) 2.65° for TM polarization as well as angular shifts of ∆θm1,TE ) 1.33° and ∆θm2,TE ) 2.0° for TE polarization are obtained when water is replaced by isopropanol. The positions of the experimentally obtained reflectivity minima were in good agreement with Fresnel calculations with the effective permittivity as the only adjustable variable © 2010 American Chemical Society

FIGURE 2. OWS R vs θ scans on PCN arrays covered by water (red circles) and isopropanol (blue squares) recorded with (a) TMpolarized light and (b) TE-polarized light. The solid lines are Fresnel fits of the experimental curves.

(solid lines in Figure 2). Notably, changes in εeff resulted in larger shifts of the reflectivity minima of the second-order modes appearing at θm2 because the amplitude of their electric field distribution is larger. Hence, higher-order modes allow sensing changes in εeff with enhanced sensitivity,7,19 and they are less damped by the Au metal layer than base modes.4,8 The achievable figure of merit FOM ) (∆θ/∆n)(1/ Γ), where Γ is the full width at half-minimum of the considered reflectivity peak, for the second-order TM mode was 196 reciprocal refractive index units (RIU-1) at λ ) 632.8 nm, thus exceeding the FOM value of 15 previously reported for surface plasmon resonance (SPR) sensors based regular angular modulation20 by more than 1 order of magnitude. FOM values similar to those obtained by OWS on PCN arrays were attained exploiting long-range surface plasmons (LRSP) propagating along opposite interfaces of extremely smooth, ultrathin gold films sandwiched between two dielectrics with similar refractive index.21 However, such configurations are prone to wear and tear. Moreover, the design of bioaffine SPR sensors may involve incorporation of functionalities that cannot easily be attached to gold surfaces. Sensor configurations based on PCNs significantly extend the range of possible chemical modifications and, consequently, the range of detectable analytes. A peculiar advantage of PCNs is the presence of surface cyanate groups that are known to readily mediate chemical coupling of peptides, proteins, and other biologically active compounds to cellulose and polysaccharides.22-24 Taurine (Mw ) 125 2175

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to the PCNs was studied by monitoring the time dependence of ∆θm2,TM for 24 h taking one measurement per second. As obvious from Figure 3b, taurine injection instantaneously led to an increase in ∆θm2,TM of ∼0.2° indicative of fast coupling of the taurine to the PCNs. Subsequently, ∆θm2,TM increased at a rate of about 0.012°/h in the subsequent slow adsorption regime, until an equilibrium value of 0.33° was reached after 17 h. Washing the PCN array 24 h after taurine injection by circulating 0.2 M NaHCO3 solution for ∼30 min through the flow cell did not significantly change ∆θm2,TM, implying that the attachment of taurine was irreversible. ⊥ || and εPCN measured by OWS were comChanges in εPCN pared to an EMT calculation that models the increase in εS related to the attachment of the taurine to the surfaces of the PCNs

⊥ εenv

FIGURE 3. (a) R vs θ scans of PCNs in 0.2 M NaHCO3 solution before (solid black circles) and 24.5 h after (open black circles) taurine injection. (b) Kinetics of taurine coupling to the PCNs tracked by changes in the angular shift of the second-order TM mode ∆θm2,TM. Injection of taurine indicated by the red arrow led to an instantaneous jump in ∆θm2,TM by 0.2°. Washing the PCN array with 0.2 M NaHCO3 solution at the point of time indicated by the green arrow did not change the equilibrated ∆θm2,TM value of 0.33°, thus indicating that taurine chemisorbed to the PCNs.

|| εenv ) εS + ftaurine(εtaurine - εS)

(2a)

(2b)

where εenv is a modified dielectric constant substituting εS in eq 2, and ftaurine is the volume fraction the adsorbed taurine molecules occupy of the volume between the PCNs. The increase in neff from 2.370 to 2.385 could be calculated from ∆θm2,TM and is related to the growth of the taurine layer. By calculating ftaurine, the thickness of the taurine layer on the PCNs was estimated to be ≈1 nm, a value indicating the formation of a dense taurine monolayer (molecular length of taurine, 0.78 nm).29 To independently confirm binding of taurine to the PCNs, we performed X-ray photoelectron spectroscopy (XPS) measurements30 on the PCN array used for the OWS measurements displayed in Figure 3 (Figure 4). Whereas the XPS spectrum of the native PCN array is featureless in the energy range from 165 to 180 eV (Figure 4, inset), the XPS spectrum of the taurine-modified PCN array showed a characteristic S 2p peak between 170 and 175 eV originating from the sulfonate group of the taurine. In conclusion, arrays of aligned polycyanurate thermoset nanorods with a length of 650 nm and a diameter of 60 nm were used as waveguiding layer in optical waveguide sensors. Waveguiding along the PCNs resulted in pronounced coupling of the guided waves to the nanorod surfaces as well as to the volume between the nanorods. Subtle changes in the effective refractive index of the PCN sensor were amplified and could be detected at high sensitivity using optical waveguide spectroscopy at visible wavelengths. The sensing figure of merit thus achieved amounted to 196 reciprocal refractive index units and is, therefore, higher than that of

Da), which is a sulfur-containing amino acid present in almost all tissues,25 was chosen as a model molecule to demonstrate surface functionalization of PCNs. The surface cyanide groups of the PCNs can react with the amino group of taurine. As a result, the surface of the thus modified PCNs bears terminal sulfonic acid (-SO3H) groups allowing for a broad range of molecule-specific detections, for additional surface modifications, as well as for layer-by-layer deposition of charged dendrimers,26 quantum dots,27 and polyelectrolytes.28 We employed OWS to monitor surface modification of PCNs with taurine. A PCN array was kept in aqueous 0.2 M NaHCO3 solution for 24 h to obtain a stable baseline prior to a R vs θ scan revealing minimum reflectivity related to the second-order TM mode at an incident angle θ ) 50.78° (Figure 3). Subsequently, 8 mM of taurine (ntaurine ) 1.48) in 10 mL of 0.2 M NaHCO3 solution was injected at a rate of 0.5 mL/min using a peristaltic pump. Constant concentration of taurine in the flow cell was maintained by cyclic flow of the taurine solution. A plot of R against θ measured 24.5 h after taurine injection and washing with 0.2 M NaHCO3 solution seen in Figure 3a reveals that the reflectivity minimum associated with the second-order TM mode was shifted from θ ) 50.78° to θ ) 51.11°. The angular shift ∆θm2,TM ) 0.33° is unambiguously indicative of the attachment of taurine to the PCNs. The kinetics of the coupling of taurine © 2010 American Chemical Society

1 (1 + ftaurine)(εtaurine - εS) 2 ) εS 1 εS + (1 - ftaurine)(εtaurine - εS) 2 εS +

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(4) (5) (6) (7) (8) (9) FIGURE 4. XPS spectrum of a taurine-modified PCN array showing a characteristic S 2p peak between 170 and 175 eV originating from the sulfonate group of the taurine. The inset shows, for comparison, a XPS spectrum of a native PCN array that is featureless in the corresponding energy range.

(10) (11) (12) (13)

other sensors based on angular modulation. The surface cyanate groups of the PCNs allow for a wide range of surface functionalizations. To illustrate, kinetics of the binding of taurine to the surface cyanate groups of the PCNs yielding PCNs with terminal sulfonic acid groups was monitored by OWS. PCN arrays may represent a versatile platform for the design of structurally flexible biosensors. For example, optical waveguide sensors could be based on arrays of nanorods with recognition sites located in their interior consisting of molecularly imprinted polymers (MIPs).

(14) (15) (16) (17) (18) (19) (20) (21) (22) (23)

Acknowledgment. We thank K. Sklarek, G. Glasser, and D. Mo¨ssner for technical support and U. Jonas for valuable discussions. Financial support from the German Research Foundation (SPP 1369) is gratefully acknowledged. H.D. thanks the European Union for the Marie Curie IntraEuropean Fellowship (MEIF-CT-2005-024731). B.Y. acknowledges financial support from the higher Education Commission (HEC) of Pakistan and Deutscher Akademischer Austauschdienst (DAAD) (#A/04/30795).

(24) (25) (26) (27) (28) (29)

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