Silica encapsulation of fluorescent nanodiamonds for colloidal stability and facile surface functionalization

Abstract

Fluorescent nanodiamonds (FNDs) emit in the near-IR and do not photobleach or photoblink. These properties make FNDs better suited for numerous imaging applications compared with commonly used fluorescence agents such as organic dyes and quantum dots. However, nanodiamonds do not form stable suspensions in aqueous buffer, are prone to aggregation, and are difficult to functionalize. Here we present a method for encapsulating nanodiamonds with silica using an innovative liposome-based encapsulation process that renders the particle surface biocompatible, stable, and readily functionalized through routine linking chemistries. Furthermore, the method selects for a desired particle size and produces a monodisperse agent. We attached biotin to the silica-coated FNDs and tracked the three-dimensional motion of a biotinylated FND tethered by a single DNA molecule with high spatial and temporal resolution.

Figure 1

(a) Nanodiamonds in a solution of TEOS are trapped in phospholipid POPC multilaminar vesicles (MLVs) that can range in size from 500–10000 nm. Ultrasonication breaks the MLVs into small unilamellar vesicles (SUVs) of nominally ~100 nm diameter. TEOS is converted into silica, catalyzed by TEA. Thereafter, free TEOS and TEA are dialyzed away. An SDS wash breaks up the liposomes to free the coated diamonds and the remaining reagents (i.e. SDS and POPC) are removed by dialysis. The final product is stabilized and monodisperse silica-encapsulated nanodiamonds. (b) Since the surface of the silica-encapsulated diamonds presents free silanol groups, a variety of silane agents can be used to attach biomolecules to the encapsulated diamonds. One such method is to conjugate a ligand to the amine group of APTES and use its remaining silanol groups to conjugate the ligand to the particle.

Figure 2

(a) Uncoated diamond in water (left vial) and silica-coated diamond in water (right vial). (b) Settling of uncoated (black circles) and silica-coated diamond (red squares) measured by light scattering. Samples were excited at 635 nm and scattering was measured at 90° relative to excitation. Settling of coated diamond was fit to a single exponential with an offset (blue line), I(t) = a ₀+a₁exp(−t/t₁). Fit parameters: a₀= 0.9784 ± 0.0006 a.u., a₁= 0.0220 ± 0.0005 a.u., t₁= 4.20 ± 0.15 hr, which we interpret as a majority (98%) stable component and a minority (2%) component that settles out slowly with a time constant of 4 hours. Error bars are the standard error of the fit. Settling of uncoated diamond was best fit to a double exponential with an offset (red line), I (t) = a₀+a₁exp(−t/t₁) +a₂exp(−t/t₂). Fit parameters: a₀= 0.1635 ± 0.0001 a.u., a₁= 0.5534 ± 0.0010 a.u., t₁= 0.7866 ± 0.0012 hr, a₂ = 0.2786 ± 0.0010 a.u., t₂= 0.2189 ± 0.0007 hr. The low values of a₀ and time constants indicate that the majority (83%) of the uncoated diamonds precipitate in less than an hour. (c) The hydrodynamic diameter (Z-avg) and (d) zeta potential of silica-coated (red) and uncoated (black) nanodiamonds as a function of pH. At pHs below the dotted lines, the absolute value of the zeta potential was less than 20 mV and flocculation with increased size was observed. Error bars are 1 standard deviation. (e) FTIR spectrum of uncoated FND. (f) FTIR spectrum of silica-coated FND. Characteristic SiO₂ bands between 800 and 1260 cm⁻¹, the Si–O band at 1090 cm⁻¹, and the Si–OH band at 950 cm⁻¹ are apparent in the spectra. In both the coated and uncoated ND samples, water was present. The band at 1635 cm⁻¹ is due to the scissor bending vibration of molecular water.

Figure 3

(a) Schematic of single-molecule binding experiment. A quartz slide passivated with biotinylated PEG was saturated with streptavidin. Biotinylated FNDs were flowed into the flow cell and specific attachment to streptavidin-biotin-PEG was probed by comparing the number of bound particles before and after washing to remove non-specifically bound particles. FNDs were excited by the evanescent field (green) in a prism-type TIRFM (Supporting Information). (b) 60% of biotinylated silica-coated FNDs and (c) 4% of non-biotinylated silica-coated FNDs remained attached to the surface after the stringent acid-base wash. (Additional information in Figure S1.)

Figure 4

Three dimensional tethered particle tracking (TPM) of DNA conformations using biotinylated FNDs. (a) One end of a 1.4 μm double-stranded DNA molecule is tethered to a quartz slide, while the other end is attached to a biotinylated ~30 nm FND. The FND is excited by the evanescent wave created at the interface of the quartz slide and buffer in the flow cell in a prism-type TIRFM. The evanescent field (green) decreases exponentially away from the quartz-buffer interface. Custom tracking software was used to track the x-, and y-positions, and the intensity ( I ) of the FND. The natural logarithm of the intensity provides a measure of the z -position as the FND moves in the exponentially varying evanescent field. An intensity threshold was used to avoid tracking errors, which resulted in missing positions of the FND at large z-extension and led to the flat tops in the distributions in (b) and (c). (b) Scatter plot of x-, y-, and z- coordinates of the tethered FND ( n = 54,263), where z = 0 denotes the maximum of the intensity, which we assume was the quartz-buffer interface. Figures (b), (c) (side view of (b)), and (d) (top view of (b) are color-coded according to r² = x² +y², where r=0 is blue and r=1 is red. (e), (f), (g), The area-normalized probability densities along x (black circles), y (black stars), and z (black diamonds) (See SI). Average DNA persistence length obtained from fits to the distributions is 55 ± 6 nm (standard deviation). Cut-off in the z-distribution corresponds to the intensity threshold used in the tracking software.