Biofunctionalization on alkylated silicon substrate surfaces via "click" chemistry

Abstract

Biofunctionalization of silicon substrates is important to the development of silicon-based biosensors and devices. Compared to conventional organosiloxane films on silicon oxide intermediate layers, organic monolayers directly bound to the nonoxidized silicon substrates via Si-C bonds enhance the sensitivity of detection and the stability against hydrolytic cleavage. Such monolayers presenting a high density of terminal alkynyl groups for bioconjugation via copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC, a "click" reaction) were reported. However, yields of the CuAAC reactions on these monolayer platforms were low. Also, the nonspecific adsorption of proteins on the resultant surfaces remained a major obstacle for many potential biological applications. Herein, we report a new type of "clickable" monolayers grown by selective, photoactivated surface hydrosilylation of α,ω-alkenynes, where the alkynyl terminal is protected with a trimethylgermanyl (TMG) group, on hydrogen-terminated silicon substrates. The TMG groups on the film are readily removed in aqueous solutions in the presence of Cu(I). Significantly, the degermanylation and the subsequent CuAAC reaction with various azides could be combined into a single step in good yields. Thus, oligo(ethylene glycol) (OEG) with an azido tag was attached to the TMG-alkyne surfaces, leading to OEG-terminated surfaces that reduced the nonspecific adsorption of protein (fibrinogen) by >98%. The CuAAC reaction could be performed in microarray format to generate arrays of mannose and biotin with varied densities on the protein-resistant OEG background. We also demonstrated that the monolayer platform could be functionalized with mannose for highly specific capturing of living targets (Escherichia coli expressing fimbriae) onto the silicon substrates.

Figure 1

Selected XPS data obtained on the films A before and after degermanylation and CuAAC reactions. XPS survey (a) and narrow scan for C1s (b, with deconvolution) of the films A, and narrow scans for Ge3d (c), Si2p (d), F1s (e) and N1s (f, with deconvolution) before (empty circle) and after (solid dot) CuAAC reaction with the CF₃-terminated azide 3.

Figure 2

Tapping mode AFM images (3×3 μm²) of the TMG-alkynyl-terminated film A before (a) and after (b) CuAAC reaction with the OEG-azide 4. The z-scale (contrast) for both images is 3 nm.

Figure 3

Progress of the CuAAC reaction on the TMG-alkynyl-terminated films A with the CF₃-terminated azide 3, monitored ex situ by the F/C ratio of the film after various reaction time in the presence (square) and absence (circle) of the ligand 10 under otherwise identical conditions: Cu(MeCN)₄PF₆ (1.25 mM), ascorbic acid (25 mM), the azide 3 (5 mM) and the ligand 10 (12.5 mM) in EtOH/H₂O 1:1 at 25°C. Each data point was obtained by reacting a film A in the reaction mixture for the given time, followed by cleaning and measuring of the F/C ratio of the film by XPS. The curve serves to guide the eyes.

Figure 4

Selected XPS narrow scans for Ge3d, N1s, and C1s of the films D and E prepared from A via CuAAC reaction with the azides 4 and 5 (Scheme 1), respectively. The data for films D include Ge3d (a, solid dots for film D vs empty circles for film A before the reaction), N1s (b, with deconvolution), N1s before (c, dots behind the squares) and after (c, squares) treatment with a 0.1% fibrinogen solution vs the N1s signal of a monolayer of fibrinogen (triangle) adsorbed on a H-Si (111) surface, and C1s (d, with deconvolution). The data for the films E include the deconvoluted C1s (e) and the N1s (f) signals.

Figure 5

Fluorescent images (a–e) of various modified surfaces incubated with fim+ and fim− E. coli, and a plot (f) of the numbers of E. coli in all images with a standard deviation on these surfaces. The combinations depicted are: the mannose-presenting film E and fim+ E. coli (a, bacterial count: 11313 ± 1241 for f), film E and fim− E. coli (b, bacterial count: 1 ± 1 for f), the glucose-presenting films F and fim+ E. coli (c, bacterial count: 7 ± 1 for f), the ethynyl-presenting films B and fim+ E. coli (d, bacterial count: 0 ± 0 for f), and film E and the fim+ E. coli that had been pre-saturated with mannose in the media (e, bacterial count: 627 ± 352 for f). Each image is representative of up to 20 images obtained on random locations at the sample surface (for examples, see Figures S2–S6 in Supporting Information).

Scheme 1

Preparation of the TMG-terminated Film (A) from the Alkenyne 2 and Its Deprotection to the Ethynyl-presenting Film B and Direct CuAAC Reactions with the Azides 3–7 Promoted by Cu⁺ and the Ligand 10 to Form Films Presenting CF₃ (C), OEG (D), Mannose (E), Glucose (F) and Biotin (G)

Scheme 2

Attachment of the Biotin-N₃ 7 and Mannose-N₃ 5 with the OEG-N₃ 4 on the TMG-alkynylterminated Films A via CuAAC Reaction, Followed by Back-filling with the OEG-N₃ 4 and Binding with FITC-labeled Avidin and Con A.