A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents.

Three different approaches to the immobilization of proteins at surfaces have been compared. All rely on the creation of surface groups that bind primary amines on lysine residues. Carboxylic acid terminated self-assembled monolayers (SAMs) have been activated using a water soluble carbodiimide to yield an active ester functionalized surface and with trifluoroacetic anhydride to yield a surface anhydride, and amine terminated SAMs have been activated using glutaraldehyde. Although the degree of surface derivatization by n-alkylamines was greater using the carbodiimide and anhydride methods under anhydrous conditions, the glutaraldehyde activation of amine terminated SAMs yielded significantly greater attachment of streptavidin than is achieved using either of the other methods. This is attributed to the susceptibility to hydrolysis of the active species formed by activation of the carboxylic acid terminated monolayers. Patterned protein structures may be formed by using both glutaraldehyde activation of amine terminated thiols and carbodiimide activation of carboxylic acid terminated thiols, in conjunction with selective photo-oxidation of oligo(ethylene glycol) terminated SAMs.

Fabrication of biomolecular nanostructures by scanning near-field photolithography of oligo(ethylene glycol)-terminated self-assembled monolayers.

The UV photo-oxidation of oligo(ethylene glycol) (OEG)-terminated self-assembled monolayers (SAMs) has been studied using static secondary ion mass spectrometry, X-ray photoelectron spectroscopy, contact angle measurement, and friction force microscopy. OEG-terminated SAMs are oxidized to yield sulfonates, but photodegradation of the OEG chain also occurs on a more rapid time scale, yielding degradation products that remain bound to the surface via gold-sulfur bonds. The oxidation of these degradation products is the rate-limiting step in the process. Photopatterning of OEG-terminated SAMs may be accomplished by using a mask and suitable light source or by using scanning near-field photolithography (SNP) in which the mask is replaced by a scanning near-field optical microscope coupled to a UV laser. Using SNP, it is possible to fabricate patterns in SAMs with a full width at half-maximum height (fwhm) as small as 9 nm, which is approximately 15 times smaller than the conventional diffraction limit. SNP-patterned OEG-terminated SAMs may be used to fabricate protein nanopatterns. By adsorbing carboxylic acid-terminated thiols into oxidized regions and converting these to active ester intermediates, it has been possible to fabricate lines of protein molecules with widths of only a few tens of nanometers.

Fabrication of biological nanostructures by scanning near-field photolithography of chloromethylphenylsiloxane monolayers.

We demonstrate the fabrication of sub-100-nm DNA surface patterns by scanning near-field optical lithography using a near-field scanning optical microscope coupled to a UV laser and a chloromethylphenylsiloxane (CMPS) self-assembled monolayer (SAM). The process involves 244-nm exposure of the CMPS SAM to create nanoscale patterns of surface carboxylic acid functional groups, followed by their conversion to the N-hydroxysuccinimidyl ester and reaction of the active ester with DNA to spatially control DNA grafting with high selectivity.