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Carbohydrate arrays fabricated on gold films were used to study carbohydrate-protein interactions with surface plasmon resonance (SPR) imaging. An immobilization scheme consisting of the formation of a surface disulfide bond was used to attach thiol-modified carbohydrates onto gold films and to fabricate carbohydrate arrays. The carbohydrate attachment steps were characterized using polarization modulation Fourier transform infrared reflection absorption spectroscopy; and poly(dimethylsiloxane) microchannels were used to immobilize probe compounds at discrete locations on a gold film. The binding of the carbohydrate-binding proteins concanavalin A (ConA) and jacalin to arrays composed of the monosaccharides mannose and galactose was monitored with SPR imaging. SPR imaging measurements were employed to accomplish the following: (i) construct adsorption isotherms for the interactions of ConA and jacalin to the carbohydrate surfaces, (ii) monitor protein binding to surfaces presenting different compositions of the immobilized carbohydrates, and (iii) measure the solution equilibrium dissociation constants for ConA and jacalin toward mannose and galactose, respectively. Adsorption coefficients (K(ADS)) of 2.2 +/- 0.8 x 10(7) M(-)(1) and 5.6 +/- 1.7 x 10(6) M(-)(1) were obtained for jacalin adsorbing to a galactose surface and ConA adsorbing to a mannose surface, respectively. The solution equilibrium dissociation (K(D)) constant for the interaction of jacalin and galactose was found to be 16 +/- 5 microM, and for ConA and mannose was found to be 200 +/- 50 microM.

A monolayer of poly(L-lysine) (PL) is attached covalently via amide bonds to an alkanethiol self-assembled monolayer (SAM) on a gold surface. The amide bond is formed in two steps: the terminal carboxylic acid groups of an 11-mercaptoundecanoic acid (MUA) SAM are first activated to the N-hydroxysulfosuccinimide (NHSS) ester, followed by reaction of this MUA-NHSS ester monolayer with the amino groups of PL to create multiple amide bond linkages to the surface. The reactivity and packing density of the MUA-NHSS esters are investigated in detail by reacting these intermediates with ammonia (NH 3 ). In the NH 3 experiments, approximately 50% of the carboxylic acids in the MUA monolayer are converted to amides during the first cycle of this two-step surface reaction. This reaction yield of 50% is limited by the steric packing of the NHSS ester intermediate. However, after three cycles of MUA activation to the NHSS ester and reaction with NH 3 , nearly all of the MUA molecules (∼80%) are converted to amides. Polarization-modulation Fourier transform infrared reflection-absorption spectroscopy (PM-FT-IRRAS) is employed to study both the NH 3 and PL reactions on the gold surface. The PM-FT-IRRAS spectrum of a covalently attached PL monolayer indicates that amide bonds are formed with the underlying MUA molecules. This conclusion is confirmed by the fact that the covalent PL monolayer resists desorption despite immersion into solutions of pH < 2 or pH > 12. Finally, the PL is derivatized with a bifunctional NHSS ester-maleimide molecule either by reaction in solution prior to covalent attachment or by reaction with PL already adsorbed to the surface. Up to 50% of the total number of lysine amino groups are converted to maleimide groups, which can be used for the subsequent attachment of sulfhydryl-containing biomolecules.The preparation of adsorption biosensors, affinity chromatography supports, enzyme-coated electrodes, and other bioanalytical devices often relies on the controlled chemical modification of surfaces. [1][2][3][4] Self-assembled monolayers (SAMs) offer a simple

Multiple microRNAs (miRNAs) are detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle-amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attomole levels.MicroRNAs (miRNAs) are small RNA molecules (19 to 23mers) that can regulate the expression of genes in plants and animals by binding to the 3′-untranslated region of messenger RNAs. 1 Several research groups have studied miRNA gene regulation in processes as diverse as cell proliferation, fat metabolism, and cell differentiation. 2,3 The recent surge of interest in miRNAs and the related small interfering RNA (siRNA) gene silencing methodology 3 has led to an increased need for the ultrasensitive detection and quantitation of small miRNAs in both solution and surface microarray formats. 4-7

The surface-sensitive optical technique of surface plasmon resonance (SPR) imaging is used to characterize ultrathin organic and biopolymer films at metal interfaces in a spatially resolved manner. Because of its high surface sensitivity and its ability to measure in real time the interaction of unlabeled biological molecules with arrays of surface-bound species, SPR imaging has the potential to become a powerful tool in biomolecular investigations. Recently, SPR imaging has been successfully implemented in the characterization of supported lipid bilayer films, the monitoring of antibody-antigen interactions at surfaces, and the study of DNA hybridization adsorption. The following is included in this review: (a) an introduction to the principles of surface plasmon resonance, (b) the details of SPR imaging instrumental design, (c) a short discussion concerning resolution, sensitivity, and quantitation in SPR imaging, (d) the details of DNA array fabrication on chemically modified gold surfaces, and (e) two examples that demonstrate the application of the SPR imaging technique to the study of protein-DNA interactions.