Control of Nanoscale Environment to Improve Stability of Immobilized Proteins on Diamond Surfaces

Abstract

Immunoassays for detection of bacterial pathogens rely on the selectivity and stability of bio-recognition elements such as antibodies tethered to sensor surfaces. The search for novel surfaces that improve the stability of biomolecules and assay performance has been pursued for a long time. However, the anticipated improvements in stability have not been realized in practice under physiological conditions because the surface functionalization layers on commonly used substrates, silica and gold, are themselves unstable on time scales of days. In this paper, we show that covalent linking of antibodies to diamond surfaces leads to substantial improvements in biological activity of proteins as measured by the ability to selectively capture cells of the pathogenic bacterium Escherichia coli O157:H7 even after exposure to buffer solutions at 37 °C for extended periods of time, approaching 2 weeks. Our results from ELISA, XPS, fluorescence microscopy, and MD simulations suggest that by using highly stable surface chemistry and controlling the nanoscale organization of the antibodies on the surface, it is possible to achieve significant improvements in biological activity and stability. Our findings can be easily extended to functionalization of micro and nanodimensional sensors and structures of biomedical diagnostic and therapeutic interest.

Figure 1

UNCD surface characterized via microscopy. a) AFM and b) SEM image of ultra nanocrystalline diamond films deposited via hot-filament chemical vapor deposition. AFM image shows that the average roughness is ~8 nm and largest peak-to-valley height is ~40 nm. SEM image shows the continuity in the diamond film at micron-scale. c) O_1s peaks obtained during XPS analysis of O-terminated, as-deposited and H-terminated UNCD films (top to bottom) show that the as-deposited UNCD films were more H-terminated than O-terminated. XPS data were obtained using an ultrahigh vacuum (P < 7 × 10⁻¹⁰ Torr) XPS system with a monochromatized Mg Kα (1253.6 eV) source (350 W, 14.0 kV) and a hemispherical analyzer equipped with a multichannel detector. H-terminated sample was produced by exposing the as-deposited samples to a 13.56 MHz inductively coupled hydrogen plasma (15 torr) for 20 minutes at 800 °C. O-terminated sample was produced by exposing the as-deposited samples to UV-lamp (254 nm, 10 mW/cm²) in air.

Figure 2

Selective capture of E.coli on antibody functionalized UNCD thin films. a) Fluorescence image of FITC-labeled antibody tethered to UNCD surface showing higher fluorescence compared to control UNCD surface. b) Fluorescence image showing labeled E.coli O157:H7 captured on antibody-UNCD surface. c) Bacteria capture density on antibody functionalized UNCD and GAPSG substrates from live isolated cultures. Fluorescently-labeled E.coli O157:H7 and K12 cells (10⁶ cfu ml⁻¹ each) were exposed to anti-O157:H7 and anti-O+K functionalized surfaces, respectively. Anti-O157:H7 functionalized surfaces were also exposed to E.coli K12 cells (10⁶ cfu ml⁻¹) to check cross-serotype reactivity. Results show similar capture and cross-serotype reactivity with antibody-tethered UNCD and GAPSG surfaces. d) Bacteria capture density for targeted and non-targeted bacteria obtained by exposing the anti-O157:H7 functionalized UNCD and GAPSG surfaces to live co-cultures of E.coli O157:H7 and Listeria monocytogenes (5 × 10⁶ cfu ml⁻₁ each). Three different blocking agents viz. casein, bovine serum albumin (BSA), and protein-free block (Pierce Scientific) were used to evaluate the effectiveness in blocking non-specific binding. Experiments in (c) and (d) were performed in triplicates.

Figure 3

Plot of E.coli O157:H7 cells captured during six regeneration and capture cycles on functionalized UNCD and GAPSG surfaces. Regeneration was performed using 0.1M glycine-HCl buffer (pH 2.1). Bacteria count after regeneration was found to coincide with readings from controls. Experiments were performed in quadruplicates.

Figure 4

E.coli O157:H7 capture on antibody-tethered UNCD and GAPSG substrates exposed to PBS at 37 °C for upto 14 days. Fluorescent images show E.coli O157:H7 captured on anti-O157:H7 functionalized UNCD and GAPSG surfaces, which were stored for 1, 5, 7, and 14 days in PBS at 37 °C. The images show the relative decrease in antibody activity on GAPSG by day 5, which was gradually also seen in case of UNCD after day 7.

Figure 5

Plot of bacteria capture density on UNCD and GAPSG surfaces a) from live E.coli O157:H7 culture (10⁶ cfu ml⁻¹) versus the number of days the antibody-tethered surfaces were exposed to PBS at 37 °C; b) from live co-culture of E.coli O157:H7 and Listeria monocytogenes (5 × 10⁵ cfu ml⁻¹, total) against the number of days the functionalized surfaces were stored in PBS at 37 °C; and c) from live E.coli O157:H7 culture (10⁶ cfu ml⁻¹) versus the number of days the antibody-tethered surfaces were exposed to PBS at 4 °C. The unusual increase in antibody activity on UNCD and GAPSG at 4 °C on day 5 and day 7, respectively, may be attributed to the favorable rearrangement of antibodies at the surface. Experiments in a, b and c were performed in triplicates.

Figure 6

a) ELISA and b) XPS results confirm presence of antibody on the UNCD and GAPSG surfaces during pro-longed exposure to PBS at 37 °C. a) Plot of optical density readings at 450 nm for quenched enzyme substrate solutions in ELISA to detect goat anti-O157:H7 tethered to UNCD and GAPSG surfaces. UNCD and GAPSG control surfaces were created by skipping the antibody functionalization step. Controls demonstrate the minimal non-specific binding during ELISA. b) Plot of N/C and N/Si atomic concentration ratios obtained via XPS on functionalized UNCD and GAPSG surfaces during the 37 °C stability tests. Nitrogen content is directly proportional to the antibody mass immobilized on the surface, assuming the nitrogen content from the self assembled layer, substrate, and contamination is negligible.

Figure 7

Water pockets on the immunosurfaces revealed by molecular dynamics (MD). a) H-terminated diamond coated with 2.6 × 10¹⁴ molecules cm⁻² aminodecane. The top panel shows a contour plot of the water density within 0.5 nm of the diamond surface, averaged over a representative 1 ns period of the 40 ns MD trajectory. The center panel shows a cut-away view of the same surface; the location of the cut away surface is indicated at the top panel as a dashed line, diamond is shown as a grey molecular surface, aminodecane as teal chains with nitrogen in blue and water as vdw spheres (oxygen in red and hydrogen in white). The bottom panel shows the potential of mean force (PMF) for bringing different types of amino acids from bulk solution to the surface. b) Water density (top), a cut away view (center) and the PMF profiles (bottom) for the silica surface coated with a 4.0 × 10¹⁴ molecules cm⁻² layer of aminopropylsilane. Silica is shown as a gray molecular surface and other molecules are shown as in panel (a). c) Water density (top), a cut away view (center) and the PMF profiles (bottom) for partially degraded the silica surface (see text). The degradation of the functionalization layer on silica creates pockets of water within the functionalization layer, which facilitates binding of amino acids as revealed by the PMF plots.

Scheme 1

Reaction steps used to attach antibody to hydrogen-terminated UNCD surface, followed by its use to capture fluorescently-labeled bacteria E.coli. a) The as-deposited hydrogen terminated UNCD film surface was photochemically grafted with trifluoroacetamide protected 10-aminodec-1-ene (TFAAD) and 1-dodecene. After attaching TFAAD to the surface (b), the amide group was deprotected in sodium borohydride (NaBH₄)-methanol solution, leaving a primary amine termination on the surface (c). The deprotected amines were further reacted with glutaraldehyde via reductive amination in sodium cyanoborohydride buffer to yield an aldehyde termination on the surface (d), which was then used to attach antibodies to the surface (e) and capture fluorescently-labeled bacteria (f).The table shows the contact angles measured on UNCD surfaces during steps (a), (b), (c), and (d).