Enhanced Photoluminescence Detection of Immunocomplex Formation by Antibody-Functionalized, Ge-Doped Biosilica from the Diatom Cyclotella sp

Abstract

Diatoms are single-celled algae that biosynthesize cell walls of biogenic silica called "frustules" that are intricately patterned at the submicron- and nanoscale. In this study, we amplified the intrinsic luminescent properties of antibody-functionalized diatom biosilica frustules for enhanced, label-free, photoluminescence (PL) detection of immunocomplex formation. It was hypothesized that metabolically doped GeO centers in antibody-functionalized diatom biosilica would enhance PL emission associated with nucleophilic immunocomplex formation. Germanium (Ge) was metabolically inserted into the frustule biosilica by two-stage cell cultivation of the centric diatom Cyclotella sp. The biosilica frustules were isolated by hydrogen peroxide treatment and thermally annealed to convert Ge oxides in the biosilica (0.4 wt% Ge) to luminescent GeO centers. The Ge-doped biosilica frustules were then functionalized with Rabbit Immunoglobulin G (IgG). Upon immunocomplex formation with its complimentary antigen goat anti-Rabbit IgG, the Ge-oxide doped, antibody-functionalized frustule biosilica increased the intensity of PL emission by a factor of 2.6 relative to immunocomplex formation by antibody-functionalized frustule biosilica without Ge. It is proposed that the luminescent GeO centers in the Ge-oxide doped frustule biosilica were more sensitive to radiative recombination than luminescent silanol groups in frustule biosilica without Ge, resulting in a higher PL emission upon immunocomplex formation.

Keywords: antibody; diatom; frustule; immunocomplex.

Figure 1

APS functionalization of diatom biosilica containing metabolically inserted Ge.

Figure 2

Two-stage cultivation of the centric diatom Cyclotella sp. for metabolic insertion of Ge-oxides. (a) Cell number density and dissolved Si vs. time profiles; (b) Si + Ge delivery and consumption during Stage II; (c) Stage II cultivation showing Ge taken up by the biomass, and Ge incorporated into the frustule after biosilica isolation, as wt% of Ge in diatom SiO₂. The first arrow at 48 h indicates when the Si + Ge feed solution in Stage IIA was turned off, the second arrow at 120 h (end of Stage IIB) indicates the biomass sample used for frustule isolation, functionalization, and PL measurements.

Figure 3

TEM images of Cyclotella frustule valve after two-stage photobioreactor cultivation, end of Stage IIB. (a,b) Control cultivation with no Ge; (c,d) Cultivation with metabolic insertion of 0.4 wt% Ge in frustule biosilica.

Figure 3

TEM images of Cyclotella frustule valve after two-stage photobioreactor cultivation, end of Stage IIB. (a,b) Control cultivation with no Ge; (c,d) Cultivation with metabolic insertion of 0.4 wt% Ge in frustule biosilica.

Figure 4

Representative photoluminescence (PL) spectra of functionalized Cyclotella diatom frustule biosilica under excitation by 337 nm laser, 2 sec integration time. (a) Frustule biosilica with no Ge; (b) Frustule biosilica with 0.4 wt% Ge. Biosilica was thermally annealed at 400 °C for 2 h prior to antibody functionalization.

Figure 5

Averaged, normalized peak PL intensity at various stages of functionalization for Cyclotella frustule biosilica vs. biosilica containing 0.4 wt% Ge. All PL emission intensities were normalized to the PL emission of the diatom biosilica frustules isolated from photobioreactor cultured cells (120 h Stage IIB) after hydrogen peroxide treatment, but before thermal annealing. Error bars represent 1.0 standard deviation based on triplicate (n = 3) sample preparations. Image on right: Epifluorescence image of Cyclotella frustule biosilica containing 0.4 wt% Ge functionalized with Rabbit IgG antibody and a fluorescein-labeled goat anti-Rabbit IgG antigen.

Figure 6

Successive PL emission enhancement of diatom frustule biosilica by metabolic insertion of Ge-oxides, amination of frustule surface, antibody functionalization, and complimentary antigen binding.