Biosensing based on self-assembled monolayers, atomic force microscopy and electrochemistry

We have developed methods to covalently immobilize biomolecules such as antibodies, antigens and oligonucleotides onto self-assembled monolayer (SAM) modified gold surfaces. Mixed SAMs were constructed and used to control the surface density of the immobilized biomolecules. Covalent immobilization methods were optimized for such immobilization. Atomic force microscopy and electrochemical methods were used to characterize the structure of SAMs and mixed SAMs, to detect biomolecule immobilization, to identify the interaction between the immobilized biomolecules and the corresponding analytes. We attempt to develop a method to identify interaction between biomolecules at the single molecule level.; Self-assembled monolayers of thioctic acid (1,2-dithiolane-3-pentanoic acid) on Au surfaces were studied by atomic force microscopy, electrochemical desorption experiments and X-ray photoelectron spectroscopy. Kinetic studies reveal a two-step adsorption process. Thioctic acid molecules are first rapidly absorbed onto the Au surface as a dithiolate; however, the thermodynamic product is an adsorbed state in which the dithiolane ring is intact.; The adsorption states of butanethiol and thioctic acid molecules in the mixed SAMs on gold surface were characterized. Scanning probe microscopy and electrochemical reductive desorption results proved that butanethiol and thioctic acid can form phase separated mixed SAMs on gold surface.; Electrochemical oxidative deposition of butanethiolate onto gold electrode from 0.5 M KOH solution was studied. Electrochemically deposited butanethiolate monolayer has the similar surface coverage as that of butanethiol SAM. It was also shown that roughly 1 electron is transferred during the first step of the oxidative deposition.; Mixed monolayers of a carboxylic acid-terminated thiol (thioctic acid, TA) and a methyl-terminated thiol (butanethiol, BT) were used in order to control the surface epitope density. The microscopic structure of the resulting antibody and antigen arrays was characterized by AFM. Individual, surface-confined rabbit IgG antibodies could be directly imaged in contact mode. The surface epitope density could be varied over approximately two orders of magnitude by changing the composition of the mixed monolayers. AFM was also used to characterize the antibody-antigen binding characteristics of these immunosurfaces, and an average binding efficiency of 22.8% was measured for rabbit IgG antibody arrays.; Preliminary results for the covalent immobilization of ssDNA oligomers onto mixed SAM and hybridization sensing are presented. (Abstract shortened by UMI.)...