Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor.

Acetylcholinesterase (AChE) inhibitors are potentially lethal but also have applications as therapeutic drugs for neurodegenerative diseases such as Alzheimer's. Enzyme inhibitor binding are difficult to be detected directly by surface plasmon resonance (SPR) due to their small molecular weight. In this article, we describe the detection of AChE inhibitor binding by SPR without the use of competitive binding or antibodies. AChE was immobilized on the gold surface of an SPR sensor through covalent attachment to a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol. The activity of the immobilized protein and the surface density were determined by using a standard photometric assay. Binding of two reversible inhibitors, which are used as therapeutic drugs, was detectable by SPR without the need to further modify the surface or the use of other reagents. The binding affinities (K(A)) obtained from the fits were 3.8 × 10(3)M(-1) for neostigmine and 1.7 × 10(3)M(-1) for eserine, showing a higher affinity of the sensor for neostigmine. We believe that the SPR sensor's ability to detect these inhibitors is due to conformational changes of the enzyme structure on inhibitor binding.

SPR-based single nucleotide mismatch biosensor.

The detection and characterization of the hybridization event of 21-base, unlabeled DNA oligonucleotides with a monolayer of complementary DNA immobilized on a gold surface, by electrochemical impedance spectroscopy and surface plasmon resonance (SPR) is presented. A thiol modification on the probe DNA strand allowed for its attachment to the surface via self-assembly. For the hybridization of full match target DNA a detection limit of 20 pM was determined. RNA hybridization was also detectable with the same sensor, with a similar detection limit. The SPR signal generated upon hybridization of the full match was always distinguishable from the single mismatch target DNA oligonucleotides when the mismatch was in the middle or at the proximal end of the target DNA sequence. However, the response of the sensor was identical for the hybridization of the full match and the distal end mismatch. The SPR sensor described is reusable over at least 20 hybridization/regeneration cycles and is insensitive to flow rate (20-800 µL min-1) or temperature (20-60 °C). Based on the SPR response, the surface density of the probe was estimated to be at least 4.3 × 1012 molecules per cm2.

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers.

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.

A multilayered approach to complex surface patterning.

A method for developing complex nanopatterns on surfaces has been developed by combining self-assembly, photolabile protecting groups, and multilayered films. An o-nitrobenzyl protecting group has been incorporated into molecular level films utilizing thiol-gold interactions. When the o-nitrobenzyl group is cleaved by ultraviolet light, a carboxylic acid terminated layer remains on the surface and is available for activation and further functionalization through amide bond formation. Using this method, multilayered films have been constructed and characterized by contact angle goniometry, cyclic voltammetry, grazing incidence infrared spectroscopy, and X-ray photoelectron spectroscopy measurements. Complex surface patterns can be achieved by creating a surface array using a photomask and then further functionalizing the irradiated area through covalent coupling. Fluorophores were attached to the deprotected regions, providing visual evidence of surface patterning using fluorescence microscopy. This approach is universal to bind moieties containing free amine groups at defined regions across a surface, allowing for the development of films with complex chemical and physicochemical properties.

Detection of oligonucleotide systematic mismatches with a surface plasmon resonance sensor.

The detection and parallel characterization of the hybridization event of 21-base, unlabeled DNA oligonucleotides with a monolayer of complementary DNA immobilized on a gold surface by surface plasmon resonance (SPR) is presented. A thiol modification on the probe DNA strand allowed for its attachment to the surface via self-assembly. For the hybridization of full match DNA a detection limit of 20 pM was determined. The change in SPR signal was always larger for the full match compared to the one-mismatch target DNA oligonucleotides when the mismatch was in the middle or at the proximal end of the target DNA. Hybridization conditions (buffer concentration, flow rate, and temperature) did not affect the ability of the sensor to discriminate for single nucleotide mismatches. To our knowledge this is the only work where a comparison on the surface hybridization efficiency is performed between proximal, distal, and middle mismatches and the effect of three hybridization parameters is studied with regard to the detection of single nucleotide mismatches using SPR.