Reserach Express@NCKU - Articles Digest

Optical sensing approach generally involves some form of fluorescence/radioactive labeling. This additional step not only increases the time and cost of the procedure, but also increases its complexity and potentially interferes with the molecular interaction by occluding binding sites or inducing conformational changes of the samples, hence leading to false negatives. Therefore, various label-free methodologies have been presented to perform biomolecular interaction analysis (BIA). Surface plasmons (SPs) are oscillations of the free electrons located on the surface of metals. When the phase velocity of an incident evanescent transverse magnetic (TM) light wave matches that of the SPs, the so-called surface plasmon resonance (SPR) phenomenon occurs, and virtually all of the incident photon energy is transferred to the SPs. SPR biosensors based on the Kretschmann configuration, which uses the attenuation total reflection (ATR) prism coupler method to excite the SPs, provide a better detection limit than other conventional methods. However, the Kretschmann configuration has a number of drawbacks, including the need for a highly sophisticated and precise metrology system and the requirement for a matching-index oil to couple the incident light from the prism into the metal surface on which the biomolecular interactions take place. Therefore, a coupled waveguide-surface plasmon resonance (CWSPR) biosensor developed in this study provides a highly sensitive and accurate detection performance. Furthermore, its simple optical setup enables the kinetics of biomolecular interactions on the sensing surface to be analyzed on an on-line basis without the need for prism coupling compared to that of a CWSPR device based on the Kretschmann configuration.

incident TM mode white light as its propagation constant,

, is near zero and lower than that of SPs, ksp:

(1)

where λ is the wavelength of the incident light and θ the incidence angle is zero for a normally incident light. However, the propagation constant of the incident light parallel to the grating surface is altered as follows:

(2)

where m is the diffraction order and Λ is the diffraction grating period. As follows from Eq. (2), the new propagation constant of the normally incident diffraction white light (θ = 0) with a subwavelength grating at m = 1 can be enhanced to match the real part of the propagation constant of SPs as Eq. (1) at a specific wavelength. In analyzing subwavelength diffraction gratings, researchers have generally employed rigorous diffraction theory (rigorous coupled wave analysis, RCWA) or effective medium theory. In RCWA, the electric field in the periodic structure is expanded as the linear combination of the spatial harmonics. Meanwhile, in the modal method, the electric field in the diffraction grating is expanded as the combination of the modes which individually satisfy the waveguide wave equation and are permitted to be expanded by the infinite spatial harmonics.

Fig. 2. Reflectivity spectra of plasmonic grating biosensor with normal incident white beam. Solid line: TM polarization with waveguide layer, Dashed line: TM polarization without waveguide layer, Dash-dot line: TE polarization with waveguide layer, Dotted line: TE polarization without waveguide layer.

Fig. 1. Schematic of coupled waveguide–surface plasmon resonance biosensor with subwavelength grating structure.

Fig. 1 presents a schematic illustration of the current CWSPR biosensor with a subwavelength grating structure. Fig. 2 compares the reflectivity spectrum of the proposed CWSPR biosensor with that of a biosensor with a similar configuration but with no waveguide layer. Fabrication of the current biosensor commenced by spinning a resist layer of polymethyl methacrylate (PMMA, MicroChem Corp.) onto a Pyrex plate of thickness 1 mm. A uni-dimensional diffraction grating measuring 100 100 μm2 was then patterned using a scanning electron microscope (SEM) equipped with a lithography system. The pattern was developed using a solution of isopropyl alcohol (IPA, Merck) and iso-butyl methyl ketone (MIBK, Merck). Subsequently, a layer of nickel was evaporated and then lifted off to create an etching mask. An anisotropic reactive ion etching process using a fluorine ion base was used to etch a subwavelength grating with a depth of 35 nm. The nickel mask was removed and a radio frequency sputtering process was used to deposit a Ta2O5 waveguide layer of thickness 285 nm and then a gold film of thickness 40 nm. The following reagents were used in the current experiments: carboxyl-terminated 16-Mercaptohexadecanoic acid (MHDA, Aldrich), N-Ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC hydrochloride, C8H17N3•HCl, M.W. = 191.70 g/mole, Fluka), N-Hydroxysuccinimide (NHS, C4H5NO3, Mw = 115.09 g/mole, Fluka), 0.5 μΜ protein G (from Streptococcus Sp., Mw = 22,600 g/mole, Sigma) dissolved into a 10 mM Tris-HCl buffer solution (pH 7.6, and 150 mM NaCl), and 1.0 μΜ anti-albumin antibody (from goat, Sigma). The surface was rinsed in deionized water, cleaned with ethanol, and dried in pure nitrogen gas. The biochip was then soaked in a 1 mM MHDA ethanol solution for 6 hours. After being soaked, the biochip was once again rinsed, cleaned and dried using deionized water, ethanol, and nitrogen, respectively, and then soaked in a 2 mM EDC 5 mM NHS solution for 12 h.

Fig. 4. Dynamic response of CWSPR during antibody interaction with protein on sensing surface.

Fig. 3. Schematic illustration of normal incident spectroscope.

In the current optical metrology system, a white light emitted from a single mode optical fiber is collimated into the microscope objective lens (4 , NA = 0.1) and focused on the sensing surface with a spot size of less than 100 μm. (See Fig. 3.) During the current molecular interaction detection processes, the specimens were pumped at a constant flow rate of 85 μl/min into a reaction cell maintained at a temperature of 27±0.1 °C. For biomolecular immobilization on the gold sensing surface, the 1 mM MHDA ethanol solution was immobilized on the gold film to form a self-assembled monolayer of the corresponding thiolate (after approximately 6 h). The biosensor was then soaked in the 2 mM EDC 5 mM NHS solution for 12 hours. Fig. 4 illustrates the variation of the resonance wavelength over the course of the dynamic response process of the thiol-modified sensor. After 212 minutes, Tris-HCl buffer was injected to wash away the nonspecific bindings, causing the resonance wavelength shift to fall to 5.7 nm. Although the optical measurement system and the proposed device are quite sound, we found some problems in the practical use. To excite SPs in a metallic film via subwavelength grating enhancement, it is preferable to use highly collimated white light as a continuum of in-plane wave vectors. However, a highly collimated white light is hard to be narrowed down to the very small sensing area ( 0.1×0.1 mm²) by the SEM lithography system. Therefore, the incident white beam contains many wave vectors that will excite secondary and tertiary SPs. This leads to the reflectivity spectrum broadened to decrease the measurement resolution. In addition, to develop the subwavelength grating structure is with an expensive lithography process. However, as designs are finalized, ordinary semiconductor manufacturing or new nanoimprinting techniques will be employed allowing extremely low cost devices to be fabricated.

In the summary, the CWSPR biosensor with a subwavelength grating structure proposed in this study provides a feasible and straightforward optical sensing platform for performing biomolecular interaction analysis in real time. The optical metrology system proposed in this study is more straightforward than the Kretschmann ATR configuration and is less sensitive to slight variations in the angle of the incident light.