Photopatterned thiol surfaces for biomolecule immobilization

Abstract

The ability to pattern small molecules and proteins on artificial surfaces is of importance for the development of new tools including tissue engineering, cell-based drug screening, and cell-based sensors. We describe here a novel "caged" thiol-mediated strategy for the fabrication of planar substrates patterned with biomolecules using photolithography. A thiol-bearing phosphoramidite (3-(2'-nitrobenzyl)thiopropyl (NBTP) phosphoramidite) was synthesized and coupled to a hydroxyl-terminated amorphous carbon substrate. A biocompatible oligo(ethylene glycol) spacer was used to resist nonspecific adsorption of protein and DNA and enhance flexibility of attached biomolecules. Thiol functionalities are revealed by UV irradiation of NBTP-modified surfaces. Both the surface coupling and photodeprotection were monitored by Polarization Modulation Fourier Transform Infrared Reflection Absorption Spectroscopy (PM-FTIRRAS) and X-ray Photoelectron Spectroscopy (XPS) measurements. The newly exposed thiols are chemically very active and react readily with a wide variety of groups. A series of molecules including biotin, DNA, and proteins were attached to the surfaces with retention of their biological activities, demonstrating the utility and generality of the approach.

Figure 1

IR spectra of (A) tetraethylene glycol functionalized, (B) NBTP protected, (C) UV deprotected amorphous carbon surfaces. Dashed vertical lines correspond to the peak frequencies listed in Table 1.

Figure 2

XPS survey spectra of (A) tetraethylene glycol functionalized, (B) NBTP protected, (C) UV deprotected amorphous carbon surfaces.

Figure 3

Variation in the intensity of NO₂ and P=O stretches of the IR spectrum as a function of phosphoramidite coupling time. 0 min corresponds to (EG)₄ modified surfaces with no phosphoramidite treatment.

Figure 4

High-resolution XPS spectra of NBTP protected and UV deprotected amorphous carbon surfaces: (A) C1s spectrum of NBTP protected carbon surface; (B) C1s spectrum of UV deprotected carbon surface; (C) N1s spectrum of NBTP protected carbon surface; (D) N1s spectrum of UV deprotected carbon surface.

Figure 5

Variation in the N1s region of the XPS spectra (A) and in the ratio of the two nitrogen components (N (399.9eV) and N (406.0eV)) to C (286.4eV) (B) as a function of UV exposure time at a wavelength of 365 nm. The UV irradiation dose is linearly proportional to exposure time, with a conversion factor of 0.09 J/(cm²·s). The data for ratio of N (406.0eV)/C (286.4eV) were fit to the first-order exponential shown in the figure (solid line). The rate constant of 0.0045 s⁻¹ corresponds to a half-life of 154 s.

Figure 6

Immobilization and detachment of ssDNA monitored by hybridization of fluorescently labeled complement. (A) Fluorescence image of an amorphous carbon substrate after ssDNA immobilization via disulfide linkages followed by hybridization of its fluorescent complement; (B) fluorescence image of the substrate after DTT reduction followed by a second round of hybridization.

Figure 7

Fluorescence image of an amorphous carbon substrate after biotin immobilization via maleimide-thiol reaction followed by staining with fluorescently-tagged avidin.

Figure 8

Fluorescence image of an amorphous carbon substrate after biotin immobilization via thioesterification followed by staining with fluorescently-tagged avidin.

Figure 9

Fluorescence images of biotinylated amorphous carbon substrates after successive incubation with NeutrAvidin, biotinylated Anti-UEA antibody or biotinylated AntiWGA antibody, and the fluorescently labeled lectins UEA or WGA.

Scheme 1

Synthesis of 3-(2’-nitrobenzyl)thiopropyl phosphoramidite. Reagents and conditions: (a) 3-mercaptopropanol, K₂CO₃, acetone, 80°C, 10 h, 75%; (b) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, diisopropylethylamine, acetonitrile, room temperature, overnight, 42%

Scheme 2

Schematic illustration showing the formation of photoactivatable substrates. Reagents and conditions: (c) tetraethyleneglycol monoallylether, 254 nm hv, 12 h; (d) 3-(2’-nitrobenzyl)thiopropyl phosphoramidite, phosphoramidite activator, 30 min; (e) 0.02 M I₂ in THF/Pyidine/H₂O, 3 min; (f) 365 nm hv, 10 min.

Scheme 3

Schematic illustration showing the immobilization of DNA via disulfide bonds (A) and biotin via thioether bonds (B). Reagents and conditions: (g) 34 mer 3’-HS-(CH₂)₃-ssDNA; (h) 19 mer complementary 3’-fluorescein-ssDNA; (i) N-Biotinoyl-N′-(6-maleimidohexanoyl)hydrazide.

Scheme 4

Schematic illustration showing the immobilization of biotin via thioesterification. Reagents and conditions: (j) EDC, DMAP, DMF, room temperature, 2 h; (k) 365 nm hv, activated biotin in DMF as exposure solvent.