Biomolecule patterning on analytical devices: a microfabrication-compatible approach.

The present work describes a methodology for patterning biomolecules on silicon-based analytical devices that reconciles 3-D biological functionalization with standard resist lift-off techniques. Unlike classic sol-gel approaches in which the biomolecule of interest is introduced within the sol mixture, a two-stage scenario has been developed. It consists first of patterning micrometer/submicrometer polycondensate scaffold structures, using classic microfabrication tools, that are then loaded with native biomolecules via a second simple incubation step under biologically friendly environmental conditions. The common compatibility issue between the biological and microfabrication worlds has been circumvented because native recognition biomolecules can be introduced into the host scaffold downstream from all compatibility issues. The scaffold can be generated on any silicon substrate via the polycondensation of aminosilane, namely, aminopropyltriethoxy silane (APTES), under conditions that are fully compatible with resist mask lithography. The scaffold porosity and high primary amine content allow proteins and nucleic acid sequences to penetrate the polycondensate and to interact strongly, thus giving rise to micrometer/submicrometer 3-D structures exhibiting high biological activity. The integration of such a biopatterning approach in the microfabrication process of silicon analytical devices has been demonstrated via the successful completion of immunoassays and nucleic acid assays.

Chemical introduction of disulfide groups on glycoproteins: a direct protein anchoring scenario.

The present work reports a direct glycoprotein immobilization protocol where the protein is chemically modified with disulfide groups which act as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The specificity of the chemical reaction, for disulfide introduction, toward carbohydrate moieties prevents any cross-reaction with other functional groups present in the protein structure. Horseradish peroxidase (HRP) was chosen as a model glycoprotein, and a biologically active densely packed SAM was obtained on gold, as demonstrated by spectrophotometry and surface plasmon resonance (SPR) spectroscopy. A hydrogen peroxide amperometric biosensor was designed using a freely diffusing mediator which exhibited high sensitivity (196 mA M-1 cm-2) and low apparent Michaelis-Menten constant (67 microM). By extension, a mixed bienzymatic monolayer, obtained by simultaneous cochemisorption of modified HRP and glucose oxidase (GOD), on a clean gold electrode displayed a high sensitivity toward glucose (13 mA M-1 cm-2). Far from competing with the versatility of the classic SAM scenario or the precision of genetic engineering, this work presents a rational and particularly rapid approach where the selectivity of chemical reactions takes advantage of the specific location of carbohydrates on glycosylated protein and antibody structures for creating highly active biological interfaces directly chemisorbed onto bare gold detection devices.

The first monoclonal antibody-based, matrix-resistant immunoassay for the carbamate herbicide asulam, in water.

To date, no ligand binding assay has been described for the carbamate herbicide asulam, although a variety of physical methods, dependent on pre-concentration of water samples, have been documented for its assessment. However, asulam is increasingly used in sensitive agricultural areas, and statutory regulations concerning its monitoring will undoubtedly become more stringent. Antibodies are optimal partners in ligand binding assays, but it is commonly understood by immunological researchers that where no antibody reactive with a particular antigen has yet been described, the immunogenicity of the antigen may be particularly restricted. By the expedient of employing a specialised approach to final immunisation with an asulam-protein conjugate, prior to the immortalisation of a specific anti-asulam antibody-producing cell, we have succeeded in generating a monoclonal antibody reactive specifically with asulam that can be configured in a convenient immunoassay. This antibody may be used flexibly in a number of ways: small sample volumes of 10 microl can be assessed to sensitivities of 4.35 x 10(-7) M (10 microg L(-1)) while avoiding discrepancies contributed by the assay matrix; this antibody-based assay can also be formatted to deliver sensitivities at levels stipulated by regulatory authorities (e.g., 4.35 x 10(-9) M or 0.1 microg L(-1)) directly from a water sample, without prior pre-concentration.