Fluorescent Aptamer Immobilization on Inverse Colloidal Crystals

Abstract

In this paper, we described a versatile two steps approach for the realization of silica inverse opals functionalized with DNA-aptamers labelled with Cy3 fluorophore. The co-assembly method was successfully employed for the realization of high quality inverse silica opal, whilst the inverse network was functionalized via epoxy chemistry. Morphological and optical assessment revealed the presence of large ordered domains with a transmission band gap depth of 32%, after the functionalization procedure. Finite Difference Time-Domain (FDTD) simulations confirmed the high optical quality of the inverse opal realized. Photoluminescence measurements evidenced the effective immobilization of DNA-aptamer molecules labelled with Cy3 throughout the entire sample thickness. This assumption was verified by the inhibition of the fluorescence of Cy3 fluorophore tailoring the position of the photonic band gap of the inverse opal. The modification of the fluorescence could be justified by a variation in the density of states (DOS) calculated by the Plane Wave Expansion (PWE) method. Finally, the development of the aforementioned approach could be seen as proof of the concept experiment, suggesting that this type of system may act as a suitable platform for the realization of fluorescence-based bio-sensors.

Keywords: DNA-aptamers; FDTD simulations; PWE method; band gap; co-assembly; colloidal crystal; fluorescence.

Figure 1

Sketch of the fabrication process for the realization of inverse silica opal and its functionalization with DNA-aptamer Cy3: (a) deposition of colloidal crystal film on the substrate, (b) calcinations of polystyrene template, (c) epoxy-functionalization of the colloidal film, (d) DNA-aptamer immobilization.

Figure 2

SEM image of inverse silica opal film obtained using co-assembly approach, showing high uniformity, with a diameter of the “holes” of about 370 nm.

Figure 3

Simulated reflectance spectra (a) θ_i = 0° and (b) at θ_i = 40° obtained by the FDTD Software package. Experimental reflectance spectra at (c) θ_i = 0° and (d) at θ_i = 40°, respectively. θ_i is the incident angle.

Figure 3

Simulated reflectance spectra (a) θ_i = 0° and (b) at θ_i = 40° obtained by the FDTD Software package. Experimental reflectance spectra at (c) θ_i = 0° and (d) at θ_i = 40°, respectively. θ_i is the incident angle.

Figure 4

(a) Reflectance spectrum acquired at normal incidence on the inverse silica opal, the arrows correspond to the position of the Fabry-Perot fringes considered for the estimation of the thickness. (b) Experimental values of mλ₁λ_1+m plotted as a function of 2n_eff(λ_1+m − λ₁) and the corresponding linear fit.

Figure 5

(a) Calculated band structure of the silica inverse opal. The inset is the reduced Brillouin zone. (b,c) Calculated DOS of inverse silica opal constituted by hollow spheres of about 370 nm at ϑ_i = 0° and ϑ_i = 40° to the direction Г-L, respectively.

Figure 6

DNA-Cy3 fluorescence of the inverse opal obtained using an Ar⁺ line (514.5 nm) and collecting the emission at 0° and 40° detection angles.