Functionalization of stable fluorescent nanodiamonds towards reliable detection of biomarkers for Alzheimer's disease

Abstract

Background: Stable and non-toxic fluorescent markers are gaining attention in molecular diagnostics as powerful tools for enabling long and reliable biological studies. Such markers should not only have a long half-life under several assay conditions showing no photo bleaching or blinking but also, they must allow for their conjugation or functionalization as a crucial step for numerous applications such as cellular tracking, biomarker detection and drug delivery.

Results: We report the functionalization of stable fluorescent markers based on nanodiamonds (NDs) with a bifunctional peptide. This peptide is made of a cell penetrating peptide and a six amino acids long β-sheet breaker peptide that is able to recognize amyloid β (Aβ) aggregates, a biomarker for the Alzheimer disease. Our results indicate that functionalized NDs (fNDs) are not cytotoxic and can be internalized by the cells. The fNDs allow ultrasensitive detection (at picomolar concentrations of NDs) of in vitro amyloid fibrils and amyloid aggregates in AD mice brains.

Conclusions: The fluorescence of functionalized NDs is more stable than that of fluorescent markers commonly used to stain Aβ aggregates such as Thioflavin T. These results pave the way for performing ultrasensitive and reliable detection of Aβ aggregates involved in the pathogenesis of the Alzheimer disease.

Keywords: Alzheimer’s disease; Amyloid beta peptide; Fluorescent markers; Nanodiamonds; Peptide R7-CLPFFD.

Fig. 1

Emission properties of NV color centers in diamond. a Atomic configuration of NV color defect in diamond. One nitrogen (blue) and three carbons (green) are adjacent to a vacancy site. The NV center can exist in two charge configurations, the neutrally charge NV0 center and the negatively charged NV− center. b Two level model of the electronic transitions of the NV− color center. c Emission spectrum of nanodiamonds. The spectrum shows a zero phonon line at 575 nm for the NV0 center and at 637 nm for the NV− center. Both centers show a broad phonon side band

Fig. 2

Functionalized nanodiamonds. a Bi-functional peptide composed of a cell penetrating R7 peptide (blue dotted area) that enhances the cellular uptake of its cargo, and a β-sheet breaker CLPFFD peptide (red dotted area) that recognizes toxic Aβ aggregates present in AD. b Zeta potential (Zp), hydrodynamic diameter (HD) and Polydispersity index (PDI) of naked and functionalized NDs. c Illustration and properties of fNDs

Fig. 3

Cell internalization of functionalized NDs in a fibroblast cell line. a Merged image of fibroblast cells with Alexa 488 labeled tubulin excited with 488 nm laser illumination (green) and fNDs excited with 532 nm illumination (red). In both cases, the emission was recorded using an avalanche photon detector (APD). b Fluorescence spectrum of nanodiamonds showing the characteristic zero phonon lines at 637 and 575 nm. c Fluorescence spectrum of Alexa 488

Fig. 4

Cell internalization of functionalized NDs in the bEnd.3 cell line. Image of bEnd.3 cells incubated with ND (panels a and c) and fND (panels b and d) at concentrations of 2 pM (panels a and b) and 20 pM (panels c and d) for 6 h

Fig. 5

Characterization of the photo stability of diamond-based color marker and Alexa Fluor 555-conjugated. a Fluorescence traces under continuous 532 nm wavelength laser illumination of Alexa Fluor 555-conjugated (green marker) and fNDs containing nitrogen-vacancy color centers (red marker) for several laser powers. b Decay rate of Alexa Fluor 555-conjugated versus the excitation power. The fluorescence decreases its intensity at a rate of 0.8 Hz/mW whereas that of the fNDs remained steady. c Fluorescence intensity vs. time illumination of Alexa Fluor 488 and (d) FITC

Fig. 6

The functionalization of NDs does not affect cell viability. Cell viability measurements evaluated with the MTS reduction assay in a HT22 and c C3 10T1/2 cell lines incubated with different concentrations of fNDs (black bars) for 24 h and non-functionalized NDs (grey bars). Further tests under higher concentrations of non-functionalized nanodiamonds were performed for b HT22 and d C3 10T1/2 cell lines. Values correspond to the mean percentage of viable cells with respect to the control cells (white bars). Error bars indicate standard deviation estimated from three experiments each carried out in triplicate

Fig. 7

Association of fNDs with Aβ fibers and plaques. a STEM image showing fibers of Aβ and fNDs together (yellow arrows show two specific fNDs, as examples). Regions without fibers show almost no fNDs. b The image shows the hippocampus of AD mouse brain tissue slides stained for detection of Aβ plaques with the anti Aβ antibody 4G8 and Alexa 488 (green) secondary antibody; and zoomed images showing fNDs illuminated with 532 nm laser. The first inset shows a confocal image zoom of 50 × 50 um2 near an Aβ plaque. The second inset shows a confocal image zoom 5 × 5 um2. Finally, a typical emission spectrum of a fND detected under 532 nm excitation near an Aβ plaque is shown